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Sample records for increases tyrosine hydroxylase

  1. Genetics Home Reference: tyrosine hydroxylase deficiency

    Science.gov (United States)

    ... Email Facebook Twitter Home Health Conditions TH deficiency Tyrosine hydroxylase deficiency Printable PDF Open All Close All ... Javascript to view the expand/collapse boxes. Description Tyrosine hydroxylase (TH) deficiency is a disorder that primarily ...

  2. Identification and optimization of tyrosine hydroxylase activity in Mucuna pruriens DC. var. utilis.

    Science.gov (United States)

    Luthra, Pratibha Mehta; Singh, Satendra

    2010-05-01

    Tyrosine hydroxylase, an iron containing tetrahydrobiopterin dependent monooxygenase (tyrosine 3-monooxygenase; EC 1.14.16.2), catalyzes the rate-limiting step in which L: -dopa is formed from the substrate L-tyrosine. L-Dopa concentration and activity of L-tyrosine hydroxylase enzyme were measured in roots, stem, leaves, pods, and immature seeds of Mucuna pruriens. Immature seeds contained maximum L-dopa content and mature leaves possessed maximum catalytic activity of tyrosine hydroxylase. Tyrosine hydroxylase from leaf homogenate was characterized as a 55 kDa protein by SDS-PAGE and Western-blot analysis with monoclonal mouse IgG2a tyrosine hydroxylase antibody. The conditions for maximum tyrosine hydroxylase activity from the leaf extract were optimized with respect to temperature, pH, cofactor 6-MPH(4), and divalent metal ions. The tyrosine hydroxylase from leaf extract possessed a K (m) value of 808.63 microM for L-tyrosine at 37 degrees C and pH 6.0. The activity of the enzyme was slightly inhibited at 2,000 microM L-tyrosine. Higher concentrations of the cofactor 6-MPH(4), however, completely inhibited the synthesis of L-dopa. Tyrosine hydroxylase converted specific monophenols such as L-tyrosine (808.63 microM) and tyramine (K (m) 1.1 mM) to diphenols L-dopa and dopamine, respectively. Fe(II) activated the enzyme while higher concentration of other divalent metals reduced its activity. For the first time, tyrosine hydroxylase from M. pruriens is being reported in this study.

  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. Role of ascorbic acid on tyrosine hydroxylase activity in the adrenal gland of guinea pig

    International Nuclear Information System (INIS)

    Nakashima, Yoko; Sanada, Hiroo; Suzue, Ryokuero; Kawada, Shoji

    1976-01-01

    The decrease of tyrosine hydroxylase activity in adrenal homogenate in scurvy was recovered after the administration of ascorbic acid. The causes of the increase in the enzyme activity after the administration of ascorbic acid have been studied. 1. No significant elevation in the enzyme activity was observed after the administration of reserpine to the scorbutic guinea pig. 2. A dose of metal chelating agent, α, α'-dipyridyl, prevented the ascorbic acid-induced or reserpine-induced increase in enzyme activity in the scorbutic and the nonscorbutic guinea pigs, respectively. 3. Tyrosine hydroxylase activity was partially recovered by the administration of FeSO 4 to the scorbutic guinea pig. From these results, it became clear that the induction of tyrosine hydroxylase which was not observed in scurvy was due to the deficiency of Fe 2+ . These results suggested that ascorbic acid affected the induction of this enzyme via Fe 2+ . (auth.)

  5. Role of ascorbic acid on tyrosine hydroxylase activity in the adrenal gland of guinea pig

    Energy Technology Data Exchange (ETDEWEB)

    Nakashima, Y; Sanada, H; Suzue, R; Kawada, S [National Inst. of Nutrition, Tokyo (Japan)

    1976-10-01

    The decrease of tyrosine hydroxylase activity in adrenal homogenate in scurvy was recovered after the administration of ascorbic acid. The causes of the increase in the enzyme activity after the administration of ascorbic acid have been studied. 1. No significant elevation in the enzyme activity was observed after the administration of reserpine to the scorbutic guinea pig. 2. A dose of metal chelating agent, ..cap alpha.., ..cap alpha..'-dipyridyl, prevented the ascorbic acid-induced or reserpine-induced increase in enzyme activity in the scorbutic and the nonscorbutic guinea pigs, respectively. 3. Tyrosine hydroxylase activity was partially recovered by the administration of FeSO/sub 4/ to the scorbutic guinea pig. From these results, it became clear that the induction of tyrosine hydroxylase which was not observed in scurvy was due to the deficiency of Fe/sup 2 +/. These results suggested that ascorbic acid affected the induction of this enzyme via Fe/sup 2 +/.

  6. 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.

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

    Science.gov (United States)

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

    2015-10-01

    Tyrosine hydroxylase catalyses the hydroxylation of L-tyrosine to l-DOPA, the rate-limiting step in the synthesis of catecholamines. Mutations in the TH gene encoding tyrosine hydroxylase are associated with the autosomal recessive disorder tyrosine hydroxylase deficiency, which manifests phenotypes varying from infantile parkinsonism and DOPA-responsive dystonia, also termed type A, to complex encephalopathy with perinatal onset, termed type B. We generated homozygous Th knock-in mice with the mutation Th-p.R203H, equivalent to the most recurrent human mutation associated with type B tyrosine hydroxylase deficiency (TH-p.R233H), often unresponsive to l-DOPA treatment. The Th knock-in mice showed normal survival and food intake, but hypotension, hypokinesia, reduced motor coordination, wide-based gate and catalepsy. This phenotype was associated with a gradual loss of central catecholamines and the serious manifestations of motor impairment presented diurnal fluctuation but did not improve with standard l-DOPA treatment. The mutant tyrosine hydroxylase enzyme was unstable and exhibited deficient stabilization by catecholamines, leading to decline of brain tyrosine hydroxylase-immunoreactivity in the Th knock-in mice. In fact the substantia nigra presented an almost normal level of mutant tyrosine hydroxylase protein but distinct absence of the enzyme was observed in the striatum, indicating a mutation-associated mislocalization of tyrosine hydroxylase in the nigrostriatal pathway. This hypomorphic mouse model thus provides understanding on pathomechanisms in type B tyrosine hydroxylase deficiency and a platform for the evaluation of novel therapeutics for movement disorders with loss of dopaminergic input to the striatum. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. 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

  9. Effects of methamphetamine exposure on anxiety-like behavior in the open field test, corticosterone, and hippocampal tyrosine hydroxylase in adolescent and adult mice.

    Science.gov (United States)

    Struntz, Katelyn H; Siegel, Jessica A

    2018-08-01

    Methamphetamine (MA) is a psychomotor stimulant drug that can alter behavior, the stress response system, and the dopaminergic system. The effects of MA can be modulated by age, however relatively little research has examined the acute effects of MA in adolescents and how the effects compare to those found in adults. The hippocampal dopamine system is altered by MA exposure and can modulate anxiety-like behavior, but the effects of MA on the hippocampal dopamine system have not been well studied, especially in adolescent animals. In order to assess potential age differences in the effects of MA exposure, this research examined the effects of acute MA exposure on locomotor and anxiety-like behavior in the open field test, plasma corticosterone levels, and hippocampal total tyrosine hydroxylase and phosphorylated tyrosine hydroxylase levels in adolescent and adult male C57BL/6 J mice. Tyrosine hydroxylase is the rate limiting enzyme in the synthesis of dopamine and was used as a marker of the hippocampal dopaminergic system. Mice were exposed to saline or 4 mg/kg MA and locomotor and anxiety-like behavior were measured in the open field test. Serum and brains were collected immediately after testing and plasma corticosterone and hippocampal total tyrosine hydroxylase and phosphorylated tyrosine hydroxylase levels measured. MA-exposed mice showed increased locomotor activity and anxiety-like behavior in the open field test compared with saline controls, regardless of age. There was no effect of MA on plasma corticosterone levels or hippocampal total tyrosine hydroxylase or phosphorylated tyrosine hydroxylase levels in either adolescent or adult mice. These data suggest that acute MA exposure during adolescence and adulthood increases locomotor activity and anxiety-like behavior but does not alter plasma corticosterone levels or hippocampal total tyrosine hydroxylase or phosphorylated tyrosine hydroxylase levels, and that these effects are not modulated by age

  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

    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...

  11. 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......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...

  12. Novel Mutations in the Tyrosine Hydroxylase Gene in the First Czech Patient with Tyrosine Hydroxylase Deficiency

    Directory of Open Access Journals (Sweden)

    K. Szentiványi

    2012-01-01

    Full Text Available Tyrosine hydroxylase deficiency manifests mainly in early childhood and includes two clinical phenotypes: an infantile progressive hypokinetic-rigid syndrome with dystonia (type A and a neonatal complex encephalopathy (type B. The biochemical diagnostics is exclusively based on the quantitative determination of the neurotransmitters or their metabolites in cerebrospinal fluid (CSF. The implementation of neurotransmitter analysis in clinical praxis is necessary for early diagnosis and adequate treatment. Neurotransmitter metabolites in CSF were analyzed in 82 children (at the age 1 month to 17 years with clinical suspicion for neurometabolic disorders using high performance liquid chromatography (HPLC with electrochemical detection. The CSF level of homovanillic acid (HVA was markedly decreased in three children (64, 79 and 94 nmol/l in comparison to age related controls (lower limit 218–450 nmol/l. Neurological findings including severe psychomotor retardation, quadruspasticity and microcephaly accompanied with marked dystonia, excessive sweating in the first patient was compatible with the diagnosis of tyrosine hydroxylase (TH deficiency (type B and subsequent molecular analysis revealed two novel heterozygous mutations c.636A>C and c.1124G>C in the TH gene. The treatment with L-DOPA/carbidopa resulted in the improvement of dystonia. Magnetic resonance imaging studies in two other patients with microcephaly revealed postischaemic brain damage, therefore secondary HVA deficit was considered in these children. Diagnostic work-up in patients with neurometabolic disorders should include analysis of neurotransmitter metabolites in CSF.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    Highlights: ► Restricted Neuroglobin expression in the mouse retina. ► Antibody validation using Neuroglobin-null mice. ► Co-expression of Neuroglobin with Melanopsin and tyrosine hydroxylase. ► 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.

  14. 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.

  15. Increased expression of tyrosine hydroxylase immunoreactivity in paraventricular and supraoptic neurons in illnesses with prolonged osmotic or nonosmotic stimulation of vasopressin release

    NARCIS (Netherlands)

    Panayotacopoulou, Maria T.; Malidelis, Yiannis I.; Fliers, Eric; Bouras, Constantin; Ravid, Rivka; Swaab, Dick F.

    2002-01-01

    Our previous studies indicated that in the human para-ventricular (PVN) and supraoptic (SON) nuclei, tyrosine hydroxylase (TH) - the first and rate-limiting enzyme in catecholamine synthesis - is localized mainly in magnocellular neurons and that antemortem factors regulate its expression. The

  16. Further RFLPs at the human tyrosine hydroxylase locus

    Energy Technology Data Exchange (ETDEWEB)

    Koerner, J; Uhlhaas, S; Propping, P; Gal, A [Institut fuer Humangenetik der Universitaet, Bonn (West Germany); Mallet, J [CNRS, Gif-sur-Yvette (France)

    1988-09-26

    The human cDNA clone (Ty7) of tyrosine hydroxylase was used. A two-allele (C1 and C2) Bg1II RFLP has been described recently with bands either at 6.9 or 8.4 kb (2). In addition, a faint invariant band appears at 9.0 kb. A third Bg1II allele (C3) with a band at 8.0 kb was detected. The allele frequency was studied in 35 and 39 unrelated Caucasians. Co-dominant inheritance for both RFLPs described here was demonstrated in 6 nuclear kindreds. RFLPs were observed under normal hybridization and wash stringencies.

  17. 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.

  18. Human phenylalanine hydroxylase is activated by H2O2: a novel mechanism for increasing the L-tyrosine supply for melanogenesis in melanocytes

    International Nuclear Information System (INIS)

    Schallreuter, Karin U.; Wazir, Umar; Kothari, Sonal; Gibbons, Nicholas C.J.; Moore, Jeremy; Wood, John M.

    2004-01-01

    Epidermal phenylalanine hydroxylase (PAH) produces L-tyrosine from the essential amino acid L-phenylalanine supporting melanogenesis in human melanocytes. Those PAH activities increase linearly in the different skin phototypes I-VI (Fitzpatrick classification) and also increase up to 24 h after UVB light with only one minimal erythemal dose. Since UVB generates also H 2 O 2 , we here asked the question whether this reactive oxygen species could influence the activity of pure recombinant human PAH. Under saturating conditions with the substrate L-phenylalanine (1 x 10 -3 M), the V max for enzyme activity increased 4-fold by H 2 O 2 (>2.0 x 10 -3 M). Lineweaver-Burk analysis identified a mixed activation mechanism involving both the regulatory and catalytic domains of PAH. Hyperchem molecular modelling and Deep View analysis support oxidation of the single Trp 120 residue to 5-OH-Trp 120 by H 2 O 2 causing a conformational change in the regulatory domain. PAH was still activated by H 2 O 2 in the presence of the electron donor/cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin despite slow oxidation of this cofactor. In vivo FT-Raman spectroscopy confirmed decreased epidermal phenylalanine in association with increased tyrosine after UVB exposure. Hence, generation of H 2 O 2 by UVB can activate epidermal PAH leading to an increased L-tyrosine pool for melanogenesis

  19. Quantitative radioautographic determination of brain tyrosine hydroxylase after direct transfer into nitro-cellulose and immunochemical detection

    International Nuclear Information System (INIS)

    Weissmann, D.; Labatut, R.; Gillon, J.Y.

    1988-01-01

    An improved quantitative immuno chemical determination of tyrosine hydroxylase brain concentrations was designed by using direct transfer into nitro-cellulose from 20 μm thick brain sections followed by immuno-detection and quantitative radioautography [fr

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

    Science.gov (United States)

    Nagatsu, T; Oka, K; Kato, T

    1979-07-21

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

  1. 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.

  2. Mobilisation of store Ca2+ activates tyrosine hydroxylase in bovine adrenal chromaffin cells

    International Nuclear Information System (INIS)

    McKenzie, S.; Marley, P.D.

    2001-01-01

    Full text: Many receptor agonists are able to activate tyrosine hydroxylase (TOH) in bovine adrenal chromaffin cells. The majority of these are dependent on extracellular Ca 2+ for this action. Entry of extracellular Ca 2+ through voltage-operated Ca 2+ channels is very effective at activating TOH. The contribution of the intracellular Ca 2+ stores to TOH activation however is not known. Previous studies have shown that mobilisation of intracellular Ca 2+ stores is effective at increasing phosphorylation of TOH, but its effect on TOH activity has not been studied. Therefore, in the present study, the effect of mobilisation of store Ca 2+ on TOH activity was investigated using primary cultures of bovine adrenal chromaffin cells. Cells were prepared from abattoir tissue and cultured for 3-6 days. TOH activity was determined over 10 minutes, measuring the 14 CO 2 produced following the hydroxylation and rapid decarboxylation of 14 C-tyrosine offered to intact cells. Caffeine increased TOH activity in a concentration-dependent manner with a maximum response of 100% increase at 20mM. This effect was not due to osmolarity since 20mM sucrose had no effect.Nor was it due to inhibition of phosphodiesterases, since the effect of caffeine was still seen in the presence of 1mM IBMX. However,caffeine-induced TOH activation was substantially reduced in the absence of extracellular Ca 2+ . The results suggest that TOH activity can be increased by mobilising intracellular Ca 2+ stores, but that this effect involves extracellular Ca 2+ influx, possibly through store-operated channels. Copyright (2001) Australian Neuroscience Society

  3. Transcriptional regulation of the tyrosine hydroxylase gene by glucocorticoid and cyclic AMP

    International Nuclear Information System (INIS)

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

    1987-01-01

    Glucocorticoid and cyclic AMP increase tyrosine hydroxylase (TH) activity and mRNA levels in pheochromocytoma cultures. The transcriptional activity of the TH gene, as measured by nuclear run-on assay, is also increased when cultures are treated with the synthetic glucocorticoid dexamethasone or agents that increase intracellular cyclic AMP, such as forskolin and 8-BrcAMP. Both inducers effect transcriptional changes within 10 min after treatment and are maximal after 30 min for forskolin and after 60 min for dexamethasone. The 5' flanking sequences of the TH gene were fused to the bacterial gene chloramphenicol acetyltransferase (CAT), and the hybrid gene was transfected into pheochromocytoma cultures and GH 4 pituitary cells. In both cell lines, a region of the TH gene containing bases -272 to +27 conferred induction of CAT by cyclic AMP, but not by glucocorticoid. The same results were found when a region of the TH gene containing -773 to + 27 was used. Thus, the sequences required for induction of TH by cyclic AMP are contained within 272 bases of 5' flanking sequence, but sequences sufficient for glucocorticoid regulation are not contained with 773 bases

  4. The expression and significance of tyrosine hydroxylase in the brain tissue of Parkinsons disease rats

    OpenAIRE

    Chen, Yuan; Lian, Yajun; Ma, Yunqing; Wu, Chuanjie; Zheng, Yake; Xie, Nanchang

    2017-01-01

    The expression and significance of tyrosine hydroxylase (TH) in brain tissue of rats with Parkinson's disease (PD) were explored and analyzed. A total of 120 clean-grade and healthy adult Wistar rats weighing 180–240 g were randomly divided equally into four groups according to the random number table method. Rats were sacrificed before and after the model establishment for 3, 6 or 8 weeks. The number of revolutions in rats was observed and the relative expression of TH mRNA in brain tissue w...

  5. 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.

  6. 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

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

    Science.gov (United States)

    Fernández-Espejo, Emilio; Bis-Humbert, Cristian

    2018-06-06

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

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

    Science.gov (United States)

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

    2016-12-01

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

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

    Science.gov (United States)

    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.

  10. 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

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

    Science.gov (United States)

    Peat, Judy; Garg, Uttam

    2016-01-01

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

  12. 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), cort...

  13. Increasing proportions of tyrosine hydroxylase-immunoreactive interneurons colocalize with choline acetyltransferase or vasoactive intestinal peptide in the developing rat cerebral cortex

    Science.gov (United States)

    Asmus, Stephen E.; Cocanougher, Benjamin T.; Allen, Donald L.; Boone, John B.; Brooks, Elizabeth A.; Hawkins, Sarah M.; Hench, Laura A.; Ijaz, Talha; Mayfield, Meredith N.

    2011-01-01

    Cortical interneurons are critical for information processing, and their dysfunction has been implicated in neurological disorders. One subset of this diverse cell population expresses tyrosine hydroxylase (TH) during postnatal rat development. Cortical TH-immunoreactive neurons appear at postnatal day (P) 16. The number of TH cells sharply increases between P16 and P20 and subsequently decreases to adult values. The absence of apoptotic markers in these cells suggests that the reduction in cell number is not due to cell death but is due to a decline in TH production. Cortical TH cells lack all additional catecholaminergic enzymes, and many coexpress GABA and calretinin, but little else is known about their phenotype or function. Because interneurons containing choline acetyltransferase (ChAT) or vasoactive intestinal peptide (VIP) share characteristics with cortical TH neurons, the coexpression of TH with ChAT or VIP was examined throughout the neocortex at P16, P20, and P30. The proportions of TH cell profiles double-labeled for ChAT or VIP significantly increased between P16 and P30. Based on their proximity to blood vessels, intrinsic cholinergic and VIPergic cells have been hypothesized to regulate cortical microcirculation. Labeling with the gliovascular marker aquaporin-4 revealed that at least half of the TH cells were apposed to microvessels at these ages, and many of these cells contained ChAT or VIP. Cortical TH neurons did not coproduce nitric oxide synthase. These results suggest that increasing proportions of cortical TH neurons express ChAT or VIP developmentally and that a subset of these TH neurons may regulate local blood flow. PMID:21295554

  14. 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.

  15. 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.)

  16. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Nobrega, J.N. [Neuroimaging Research Section, Clarke Institute of Psychiatry, Toronto (Canada); Gernert, M.; Loescher, W. [Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Buenteweg 17, D-30559 Hannover (Germany); Raymond, R.; Belej, T. [Neuroimaging Research Section, Clarke Institute of Psychiatry, Toronto (Canada); Richter, A. [Department of Pharmacology, Toxicology and Pharmacy, School of Veterinary Medicine, Buenteweg 17, D-30559 Hannover (Germany)

    1999-08-01

    Recent pharmacological studies and receptor analyses have suggested that dopamine neurotransmission is enhanced in mutant dystonic hamsters (dt{sup 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 [{sup 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 [{sup 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 [{sup 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 reserved000.

  18. 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

  19. Differential Expression of Tyrosine Hydroxylase Protein and Apoptosis-Related Genes in Differentiated and Undifferentiated SH-SY5Y Neuroblastoma Cells Treated with MPP+

    OpenAIRE

    Khwanraj, Kawinthra; Phruksaniyom, Chareerut; Madlah, Suriyat; Dharmasaroja, Permphan

    2015-01-01

    The human neuroblastoma SH-SY5Y cell line has been used as a dopaminergic cell model for Parkinson's disease research. Whether undifferentiated or differentiated SH-SY5Y cells are more suitable remains controversial. This study aims to evaluate the expression of apoptosis-related mRNAs activated by MPP+ and evaluate the differential expression of tyrosine hydroxylase (TH) in undifferentiated and retinoic acid- (RA-) induced differentiated cells. The western blot results showed a gradual decre...

  20. Characterisation of tryptic peptides of phosphorylated tyrosine hydroxylase by high-pressure liquid chromatography electrospray ionisation mass spectrometry

    International Nuclear Information System (INIS)

    Graham, Mark E.; Dickson, Phillip W.; Dunkley, Peter R.; Nagy-Felsobuki, Ellak I. von

    2005-01-01

    Tyrosine hydroxylase (TH) is involved in the biosynthesis of catecholamines and is activated by phosphorylation. Phosphorylated TH was analysed using high-pressure liquid chromatography combined with electrospray mass spectrometry (HPLC ESI-MS). Two mass scanning methods were used to detect tryptic cleavage products of TH. In the positive electrospray ionisation mode (ESI+), the peptides that contain the phosphorylation sites of TH were identified. In the alternative method, a phosphopeptide was detected in the negative electrospray ionisation mode (ESI-) using single ion monitoring in combination with a sequential ESI+ switching experiment. A raised baseline interfered with detection of hydrophilic peptides in ESI-, with the signal-to-noise ratio indicating that the method was operating near the limit of detection for a conventional electrospray source. The switching method improved the certainty of identification of phosphopeptides

  1. 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

  2. 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

  3. Tyrosine hydroxylase regulatory domain as indicator of enzyme sensitivity to irradiation

    International Nuclear Information System (INIS)

    Mustafayeva, N.N.; Alieva, I.N.; Aliev, Ds.I.

    2002-01-01

    Full text: At the present time contra dictionary and variously kind opinions concern to effect of different level of irradiation on the structure and functional activity of the tyrosine hydroxylase (TH), the key a rate-limiting enzyme in the biosynthesis of catecholamines are discussed in this study. To date, the effect of the irradiation on the both catalytic and N-terminal regulatory domains of TH localized in the different parts of the brain has been established. Th is responsible for dopamine, noradrenaline and adrenaline catecholamines neuro mediators biosynthesis, so a number of pathological changes in an organism has been induced by the structural reorganization different parts of the TH domains under pathological effect of environment. The available conformational states of the human TH type 1 (hTH1) regulatory domain, the activity of which is regulated by the feedback inhibition of the catecholamine products including dopamine has been established by the method of molecular mechanics. It is shown that N-terminal sequence Met30-Ser40 of hTH1 located between the two a-helices (residues 16-29 and residues 41-59) has a number of low-energy conformational states. The most available structures consists of b-turn type II on the pentapeptide fragment of hTH1. This fragment distortion under pathological factors effect, i.e. irradiation may lead to global reorganization in enzyme structure as well as at the enzyme catalytic and regulatory functions

  4. 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.

  5. The metabolism of L-phenylalanine and L-tyrosine by liver cells isolated from adrenalectomized rats and from streptozotocin-diabetic rats.

    OpenAIRE

    Stanley, J C; Fisher, M J; Pogson, C I

    1985-01-01

    Flux through, and maximal activities of, key enzymes of phenylalanine and tyrosine degradation were measured in liver cells prepared from adrenalectomized rats and from streptozotocin-diabetic rats. Adrenalectomy decreased the phenylalanine hydroxylase flux/activity ratio; this was restored by steroid treatment in vivo. Changes in the phosphorylation state of the hydroxylase may mediate these effects; there was no significant change in the maximal activity of the hydroxylase. Tyrosine metabol...

  6. 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.

  7. The conversion of phenylalanine to tyrosine in man. Direct measurement by continuous intravenous tracer infusions of L-[ring-2H5]phenylalanine and L-[1-13C] tyrosine in the postabsorptive state

    International Nuclear Information System (INIS)

    Clarke, J.T.; Bier, D.M.

    1982-01-01

    Steady state phenylalanine and tyrosine turnover and the rate of conversion of phenylalanine of tyrosine in vivo were determined in 6 healthy postabsorptive adult volunteers. Continuous infusions of tracer amounts of L-[ring- 2 H5]phenylalanine were determined intravenously for 13-14 hr. After 9-10 hr, a priming dose followed by a continuous infusion of L-[1- 13 C]tyrosine was added and maintained, along with the [ 2 H5]phenylalanine infusion, for 4 hr. Venous plasma samples were obtained before the initiation of each infusion and every 30 min during the course of the combined [ 2 H5]phenylalanine and [ 13 C]tyrosine infusion for determination of isotopic enrichments of [ 2 H5]phenylalanine, [ 13 C]tyrosine, and [ 2 H4]tyrosine by gas chromatograph-mass spectrometric analysis of the N-trifluoroacetyl-, methyl ester derivatives of the amino acids. Calculated from the observed enrichments, free phenylalanine and tyrosine turnover rates were 36.1 +/- 5.1 mumole . kg-1 . h-1 and 39.8 +/- 3.5 mumole . kg-1 . h-1, respectively. Phenylalanine was converted to tyrosine at the rate of 5.83 +/- 0.59 mumole . kg-1 . h-1, accounting for approximately 16% of either the phenylalanine or the tyrosine flux. The results indicate that the normal basal steady state phenylalanine hydroxylase activity in vivo in man is lower than that obtained from phenylalanine loading studies. This supports the existence of some type of substance activation of the enzyme as reflected in the previously reported exponential relationship between phenylalanine concentration and phenylalanine hydroxylase activity in vitro. The use of continuous simultaneous infusions of tracer amounts of stable isotope-labeled phenylalanine and tyrosine provides a direct means for studying physiological regulation of phenylalanine hydroxylase activity in vivo

  8. 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.

  9. 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.

  10. Transient knockdown of tyrosine hydroxylase during development has persistent effects on behaviour in adult zebrafish (Danio rerio.

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    Isabel Formella

    Full Text Available Abnormal dopamine (DA signaling is often suggested as causative in schizophrenia. The other prominent hypothesis for this disorder, largely driven by epidemiological data, is that certain adverse events during the early stages of brain development increase an individual's risk of developing schizophrenia later in life. However, the clinical and preclinical literature consistently implicates behavioural, cognitive, and pharmacological abnormalities, implying that DA signaling is abnormal in the adult brain. How can we reconcile these two major hypotheses underlying much of the clinical and basic research into schizophrenia? In this study we have transiently knocked down tyrosine hydroxylase (TH, the rate limiting enzyme in DA synthesis gene expression in the early stages of brain development in zebrafish using morpholinos. We show that by adulthood, TH and DA levels have returned to normal and basic DA-mediated behaviours, such as locomotion, are also normal. However, when they were exposed to a novel environment the levels of freezing and immediate positioning in deeper zones were significantly reduced in these adult fish. The neurochemistry underlying these behaviours is complex, and the exact mechanisms for these abnormal behaviours remains unknown. This study demonstrates that early transient alterations in DA ontogeny can produce persistent alterations in adult brain function and suggests that the zebrafish may be a promising model animal for future studies directed at clarifying the basic neurodevelopmental mechanisms behind complex psychiatric disease.

  11. Cytoplasm-predominant Pten associates with increased region-specific brain tyrosine hydroxylase and dopamine D2 receptors in mouse model with autistic traits.

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    He, Xin; Thacker, Stetson; Romigh, Todd; Yu, Qi; Frazier, Thomas W; Eng, Charis

    2015-01-01

    Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by impairment in social communication/interaction and inflexible/repetitive behavior. Several lines of evidence support genetic factors as a predominant cause of ASD. Among those autism susceptibility genes that have been identified, the PTEN tumor suppressor gene, initially identified as predisposing to Cowden heritable cancer syndrome, was found to be mutated in a subset of ASD patients with extreme macrocephaly. However, the ASD-relevant molecular mechanism mediating the effect of PTEN mutations remains elusive. We developed a Pten knock-in murine model to study the effects of Pten germline mutations, specifically altering subcellular localization, in ASD. Proteins were isolated from the hemispheres of the male littermates, and Western blots were performed to determine protein expression levels of tyrosine hydroxylase (TH). Immunohistochemical stains were carried out to validate the localization of TH and dopamine D2 receptors (D2R). PC12 cells ectopically expressing either wild-type or missense mutant PTEN were then compared for the differences in TH expression. Mice carrying Pten mutations have high TH and D2R in the striatum and prefrontal cortex. They also have increased phosphorylation of cAMP response element-binding protein (CREB) and TH. Mechanistically, PTEN downregulates TH production in PC12 cells via inhibiting the phosphoinositide 3-kinase (PI3K)/CREB signaling pathway, while PTEN reduces TH phosphorylation via suppressing MAPK pathway. Unlike wild-type PTEN but similar to the mouse knock-in mutant Pten, three naturally occurring missense mutations of PTEN that we previously identified in ASD patients, H93R, F241S, and D252G, were not able to suppress TH when overexpressed in PC12 cells. In addition, two other PTEN missense mutations, C124S (pan phosphatase dead) and G129E (lipid phosphatase dead), failed to suppress TH when ectopically expressed in PC12 cells

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

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    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.

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

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    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

  14. Lipoprotein cholesterol uptake mediates upregulation of bile acid synthesis by increasing cholesterol 7a-hydroxylase but not sterol 27- hydroxylase gene expression in cultured rat hepatocytes.

    NARCIS (Netherlands)

    Post, S.M.; Twisk, J.W.R.; van der Fits, L.T.E.; Wit, E.C.M.; Hoekman, M.F.M.; Mager, W.H.; Princen, H.M.G.

    1999-01-01

    Lipoproteins may supply substrate for the formation of bile acids, and the amount of hepatic cholesterol can regulate bile-acid synthesis and increase cholesterol 7α-hydroxylase expression. However, the effect of lipoprotein cholesterol on sterol 27-hydroxylase expression and the role of different

  15. Effects of manganese on tyrosine hydroxylase (TH) activity and TH-phosphorylation in a dopaminergic neural cell line

    International Nuclear Information System (INIS)

    Zhang Danhui; Kanthasamy, Arthi; Anantharam, Vellareddy; Kanthasamy, Anumantha

    2011-01-01

    Manganese (Mn) exposure causes manganism, a neurological disorder similar to Parkinson's disease. However, the cellular mechanism by which Mn impairs the dopaminergic neurotransmitter system remains unclear. We previously demonstrated that caspase-3-dependent proteolytic activation of protein kinase C delta (PKCδ) plays a key role in Mn-induced apoptotic cell death in dopaminergic neurons. Recently, we showed that PKCδ negatively regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, by enhancing protein phosphatase-2A activity in dopaminergic neurons. Here, we report that Mn exposure can affect the enzymatic activity of TH, the rate-limiting enzyme in dopamine synthesis, by activating PKCδ-PP2A signaling pathway in a dopaminergic cell model. Low dose Mn (3-10 μM) exposure to differentiated mesencephalic dopaminergic neuronal cells for 3 h induced a significant increase in TH activity and phosphorylation of TH-Ser40. The PKCδ specific inhibitor rottlerin did not prevent Mn-induced TH activity or TH-Ser40 phosphorylation. On the contrary, chronic exposure to 0.1-1 μM Mn for 24 h induced a dose-dependent decrease in TH activity. Interestingly, chronic Mn treatment significantly increased PKCδ kinase activity and protein phosphatase 2A (PP2A) enzyme activity. Treatment with the PKCδ inhibitor rottlerin almost completely prevented chronic Mn-induced reduction in TH activity, as well as increased PP2A activity. Neither acute nor chronic Mn exposures induced any cytotoxic cell death or altered TH protein levels. Collectively, these results demonstrate that low dose Mn exposure impairs TH activity in dopaminergic cells through activation of PKCδ and PP2A activity.

  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. The neurotropic parasite Toxoplasma gondii increases dopamine metabolism.

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    Emese Prandovszky

    Full Text Available The highly prevalent parasite Toxoplasma gondii manipulates its host's behavior. In infected rodents, the behavioral changes increase the likelihood that the parasite will be transmitted back to its definitive cat host, an essential step in completion of the parasite's life cycle. The mechanism(s responsible for behavioral changes in the host is unknown but two lines of published evidence suggest that the parasite alters neurotransmitter signal transduction: the disruption of the parasite-induced behavioral changes with medications used to treat psychiatric disease (specifically dopamine antagonists and identification of a tyrosine hydroxylase encoded in the parasite genome. In this study, infection of mammalian dopaminergic cells with T. gondii enhanced the levels of K+-induced release of dopamine several-fold, with a direct correlation between the number of infected cells and the quantity of dopamine released. Immunostaining brain sections of infected mice with dopamine antibody showed intense staining of encysted parasites. Based on these analyses, T. gondii orchestrates a significant increase in dopamine metabolism in neural cells. Tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, was also found in intracellular tissue cysts in brain tissue with antibodies specific for the parasite-encoded tyrosine hydroxylase. These observations provide a mechanism for parasite-induced behavioral changes. The observed effects on dopamine metabolism could also be relevant in interpreting reports of psychobehavioral changes in toxoplasmosis-infected humans.

  18. Abnormal tyrosine and phenylalanine metabolism in patients with tyrosyluria and phenylketonuria; gas-liquid chromatographic analysis of urinary metabolites

    NARCIS (Netherlands)

    Wadman, S.K.; Heiden, C. van der; Ketting, D.; Sprang, F.J. van

    Gas-liquid chromatographic methods have been developed for the analysis of: urinary phenylalanine metabolites (I) in patients with phenylketonuria, tyrosine metabolites (II) in patients with a disturbed tyrosine metabolism at the level of p-hydroxyphenylpyruvate hydroxylase, and homogentisic acid in

  19. 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.

  20. Differential Expression of Tyrosine Hydroxylase Protein and Apoptosis-Related Genes in Differentiated and Undifferentiated SH-SY5Y Neuroblastoma Cells Treated with MPP+

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    Kawinthra Khwanraj

    2015-01-01

    Full Text Available The human neuroblastoma SH-SY5Y cell line has been used as a dopaminergic cell model for Parkinson’s disease research. Whether undifferentiated or differentiated SH-SY5Y cells are more suitable remains controversial. This study aims to evaluate the expression of apoptosis-related mRNAs activated by MPP+ and evaluate the differential expression of tyrosine hydroxylase (TH in undifferentiated and retinoic acid- (RA- induced differentiated cells. The western blot results showed a gradual decrease in TH in undifferentiated cells and a gradual increase in TH in differentiated cells from days 4 to 10 after cell plating. Immunostaining revealed a gradual increase in TH along with neuritic outgrowth in differentiated cells on days 4 and 7 of RA treatment. For the study on cell susceptibility to MPP+ and the expression of apoptosis-related genes, MTT assay showed a decrease in cell viability to approximately 50% requiring 500 and 1000 μM of MPP+ for undifferentiated and RA-differentiated cells, respectively. Using real-time RT-PCR, treatment with 500 μM MPP+ led to significant increases in the Bax/Bcl-2 ratio, p53, and caspase-3 in undifferentiated cells but was without significance in differentiated cells. In conclusion, differentiated cells may be more suitable, and the shorter duration of RA differentiation may make the SH-SY5Y cell model more accessible.

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

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    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.

  2. Human Albumin Prevents 6-Hydroxydopamine-Induced Loss of Tyrosine Hydroxylase in In Vitro and In Vivo

    Science.gov (United States)

    Zhang, Li-Juan; Xue, Yue-Qiang; Yang, Chun; Yang, Wei-Hua; Chen, Long; Zhang, Qian-Jin; Qu, Ting-Yu; Huang, Shile; Zhao, Li-Ru; Wang, Xiao-Min; Duan, Wei-Ming

    2012-01-01

    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. PMID:22815976

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

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    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

  4. Renal denervation decreases blood pressure and renal tyrosine hydroxylase but does not augment the effect of hypotensive drugs.

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    Skrzypecki, Janusz; Gawlak, Maciej; Huc, Tomasz; Szulczyk, Paweł; Ufnal, Marcin

    2017-01-01

    The effect of renal denervation on the efficacy of antihypertensive drugs has not yet been elucidated. Twenty-week-old spontaneously hypertensive rats were treated with metoprolol, losartan, indapamide, or saline (controls) and assigned to renal denervation or a sham procedure. Acute hemodynamic measurements were performed ten days later. Series showing a significant interaction between renal denervation and the drugs were repeated with chronic telemetry measurements. In the saline series, denervated rats showed a significantly lower mean arterial blood pressure (blood pressure) than the sham-operated rats. In contrast, in the metoprolol series denervated rats showed a significantly higher blood pressure than sham rats. There were no differences in blood pressure between denervated and sham rats in the losartan and indapamide series. In chronic studies, a 4-week treatment with metoprolol caused a decrease in blood pressure. Renal denervation and sham denervation performed 10 days after the onset of metoprolol treatment did not affect blood pressure. Denervated rats showed markedly reduced renal nerve tyrosine hydroxylase levels. In conclusion, renal denervation decreases blood pressure in hypertensive rats. The hypotensive action of metoprolol, indapamide, and losartan is not augmented by renal denervation, suggesting the absence of synergy between renal denervation and the drugs investigated in this study.

  5. Effect of hyperthyroidism on circulating prolactin and hypothalamic expression of tyrosine hydroxylase, prolactin signaling cascade members and estrogen and progesterone receptors during late pregnancy and lactation in the rat.

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    Pennacchio, Gisela E; Neira, Flavia J; Soaje, Marta; Jahn, Graciela A; Valdez, Susana R

    2017-02-15

    Hyperthyroidism (HyperT) compromises pregnancy and lactation, hindering suckling-induced PRL release. We studied the effect of HyperT on hypothalamic mRNA (RT-qPCR) and protein (Western blot) expression of tyrosine hydroxylase (TH), PRL receptor (PRLR) and signaling pathway members, estrogen-α (ERα) and progesterone (PR) receptors on late pregnancy (days G19, 20 and 21) and early lactation (L2) in rats. HyperT advanced pre-partum PRL release, reduced circulating PRL on L2 and increased TH mRNA (G21 and L2), p-TH, PRLR mRNA, STAT5 protein (G19 and L2), PRLR protein (G21) and CIS protein (G19). PRs mRNAs and protein decreased on G19 but afterwards PRA mRNA (G20), PRB mRNA (G21) and PRA mRNA and protein (L2) increased. ERα protein increased on G19 and decreased on G20. Thus, the altered hypothalamic PRLR, STAT5, PR and ERα expression in hyperthyroid rats may induce elevated TH expression and activation, that consequently, elevate dopaminergic tone during lactation, blunting suckling-induced PRL release and litter growth. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  7. 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.

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

    Science.gov (United States)

    Wu, Shao Ping; Fu, Ai Ling; Wang, Yu Xia; Yu, Lei Ping; Jia, Pei Yuan; Li, Qian; Jin, Guo Zhang; Sun, Man Ji

    2006-07-21

    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 microM) 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.

  9. 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

  10. RNAi down-regulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua.

    Science.gov (United States)

    Kumar, Ritesh; Vashisth, Divya; Misra, Amita; Akhtar, Md Qussen; Jalil, Syed Uzma; Shanker, Karuna; Gupta, Madan Mohan; Rout, Prashant Kumar; Gupta, Anil Kumar; Shasany, Ajit Kumar

    2016-05-25

    Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of trans-cinnamic acid in the plant due to AaC4H knockdown was accompanied with the reduction of p-coumaric acid, total phenolics, anthocyanin, cinnamate-4-hydroxylase (C4H) and phenylalanine ammonia lyase (PAL) activities but increase in salicylic acid (SA) and artemisinin. Interestingly, feeding trans-cinnamic acid to the RNAi line increased the level of artemisinin along with benzoic (BA) and SA with no effect on the downstream metabolites p-coumaric acid, coniferylaldehyde and sinapaldehyde, whereas p-coumaric acid feeding increased the content of downstream coniferylaldehyde and sinapaldehyde with no effect on BA, SA, trans-cinnamic acid or artemisinin. SA is reported earlier to be inducing the artemisinin yield. This report demonstrates the link between the phenylpropanoid/lignin pathway with artemisinin pathway through SA, triggered by accumulation of trans-cinnamic acid because of the blockage at C4H.

  11. RNAi down-regulation of cinnamate-4-hydroxylase increases artemisinin biosynthesis in Artemisia annua

    OpenAIRE

    Kumar, Ritesh; Vashisth, Divya; Misra, Amita; Akhtar, Md Qussen; Jalil, Syed Uzma; Shanker, Karuna; Gupta, Madan Mohan; Rout, Prashant Kumar; Gupta, Anil Kumar; Shasany, Ajit Kumar

    2016-01-01

    Cinnamate-4-hydroxylase (C4H) converts trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthesis pathway. Earlier we reported increased expression of AaCYP71AV1 (an important gene of artemisinin biosynthesis pathway) caused by CA treatment in Artemisia annua. Hence, AaC4H gene was identified, cloned, characterized and silenced in A. annua with the assumption that the elevated internal CA due to knock down may increase the artemisinin yield. Accumulation of t...

  12. Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices

    International Nuclear Information System (INIS)

    Stratton, K.R.; Worley, P.F.; Huganir, R.L.; Baraban, J.M.

    1989-01-01

    The authors have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment of intact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine- and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipid system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation

  13. Chronic desipramine treatment alters tyrosine hydroxylase but not norepinephrine transporter immunoreactivity in norepinephrine axons in the rat prefrontal cortex

    Science.gov (United States)

    Erickson, Susan L.; Gandhi, Anjalika R.; Asafu-Adjei, Josephine K.; Sampson, Allan R.; Miner, LeeAnn; Blakely, Randy D.; Sesack, Susan R.

    2011-01-01

    Pharmacological blockade of norepinephrine (NE) reuptake is clinically effective in treating several mental disorders. Drugs that bind to the NE transporter (NET) alter both protein levels and activity of NET and also the catecholamine synthetic enzyme tyrosine hydroxylase (TH). We examined the rat prefrontal cortex (PFC) by electron microscopy to determine whether the density and subcellular distribution of immunolabeling for NET and colocalization of NET with TH within individual NE axons were altered by chronic treatment with the selective NE uptake inhibitor desipramine (DMI). Following DMI treatment (21 days, 15 mg/kg/day), NET-immunoreactive (-ir) axons were significantly less likely to colocalize TH. This finding is consistent with reports of reduced TH levels and activity in the locus coeruleus after chronic DMI and indicates a reduction of NE synthetic capacity in the PFC. Measures of NET expression and membrane localization, including the number of NET-ir profiles per tissue area sampled, the number of gold particles per NET-ir profile area, and the proportion of gold particles associated with the plasma membrane, were similar in DMI and vehicle treated rats. These findings were verified using two different antibodies directed against distinct epitopes of the NET protein. The results suggest that chronic DMI treatment does not reduce NET expression within individual NE axons in vivo or induce an overall translocation of NET protein away from the plasma membrane in the PFC as measured by ultrastructural immunogold labeling. Our findings encourage consideration of possible postranslational mechanisms for regulating NET activity in antidepressant-induced modulation of NE clearance. PMID:21208501

  14. Tyrosine content, influx and accumulation rate, and catecholamine biosynthesis measured in vivo, in the central nervous system and in peripheral organs of the young rat. Influence of neonatal hypo- and hyperthyroidism.

    Science.gov (United States)

    Diarra, A; Lefauconnier, J M; Valens, M; Georges, P; Gripois, D

    1989-10-01

    The influence of neonatal hypo- and hyperthyroidism on different aspects of tyrosine metabolism in the hypothalamus, striatum, brainstem, adrenal glands, heart and brown adipose tissue (BAT) were studied in 14-day old rats. The synthesis rate of catecholamines (CA) was also determined in vivo after the injection of labelled tyrosine. Hypothyroidism increases tyrosinaemia and endogenous tyrosine concentration in the hypothalamus and BAT. Hyperthyroidism decreases tyrosinaemia and endogenous tyrosine levels in the striatum, adrenals and heart. The accumulation rate of tyrosine determined 30 min after an intravenous injection of the labelled amino acid has been determined in the organs, together with the influx of the amino acid, determined within 20s. Hypothyroidism increases tyrosine accumulation rate in all the organs studied, and tyrosine clearance is decreased in the striatum and brainstem; together with an increased tyrosinaemia, this leads to a normal influx. The influx of tyrosine is increased in the hypothalamus. Hyperthyroidism decreases tyrosine accumulation rate in all the organs except the adrenals. These results indicate that the thyroid status of the young rat can influence tyrosine uptake mechanisms, without modifying an organ's tyrosine content. The fact that hypothyroidism increases tyrosine influx in the hypothalamus without modifying it in the brainstem and striatum reflects an heterogeneous reactivity to the lack of thyroid hormones in different brain structures. Neonatal hypothyroidism decreases the CA synthesis rate in the striatum, the heart and the interscapular brown adipose tissue, while synthesis was enhanced in the brainstem and the adrenals. It is likely that these variations in CA synthesis are due to thyroid hormone modulation of tyrosine hydroxylase activity, the enzyme which catalyses the rate limiting step in CA biosynthesis.

  15. 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.

  16.   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

    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...... show effective therapeutic vaccination against neuroblastoma with a novel rationally designed TH minigene vaccine without induction of autoimmunity providing an important baseline for future clinical application of this strategy....

  17. 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.

  18. Tyrosine Hydroxylase (TH)- and Aromatic-L-Amino Acid Decarboxylase (AADC)-Immunoreactive Neurons of the Common Marmoset (Callithrix jacchus) Brain: An Immunohistochemical Analysis

    Science.gov (United States)

    Karasawa, Nobuyuki; Hayashi, Motoharu; Yamada, Keiki; Nagatsu, Ikuko; Iwasa, Mineo; Takeuchi, Terumi; Uematsu, Mitsutoshi; Watanabe, Kazuko; Onozuka, Minoru

    2007-01-01

    From the perspective of comparative morphology, the distribution of non-monoaminergic neurons in the common marmoset (Callithrix jacchus) was investigated using an immunohistochemical method with specific antibodies to tyrosine hydroxylase (TH) and aromatic-L-amino acid decarboxylase (AADC). TH-immunoreactive (IR) neurons (but not AADC-IR) neurons were observed in the olfactory tubercle, preoptic suprachiasmatic nucleus, periventricular hypothalamic nucleus, arcuate nucleus, paraventricular nucleus, periaqueductal gray matter, medial longitudinal fasciculus, substantia nigra, and nucleus solitaris. In contrast, AADC-IR (but not TH-IR), small, oval and spindle-shaped neurons were sparsely distributed in the following areas: the hypothalamus from the anterior nucleus to the lateral nucleus, the dorsomedial nucleus, the dorsomedial area of the medial mammillary nucleus and the arcuate nucleus; the midbrain, including the stria medullaris and substantia nigra; and the medulla oblongata, including the dorsal area of the nucleus solitaris and the medullary reticular nucleus. The distribution of AADC-IR neurons was not as extensive in the marmoset as it is in rats. However, these neurons were located in the marmoset, but not the rat substantia nigra. Furthermore, AADC-IR neurons that are present in the human striatum were absent in that of the marmoset. The present results indicate that the distribution of non-monoaminergic neurons in the brain of the common marmoset is unique and different from that in humans and rodents. PMID:17653300

  19. Presence of corticotrophin-releasing factor and/or tyrosine hydroxylase in cells of a neural brain-testicular pathway that are labelled by a transganglionic tracer.

    Science.gov (United States)

    James, P; Rivier, C; Lee, S

    2008-02-01

    Our laboratory has shown that male testosterone levels are not solely controlled by the release of hypothalamic gonadotrophin-releasing hormone and pituitary luteinising hormone, but are also regulated by a multisynaptic pathway connecting the brain and the testis that interferes with the testosterone response to gonadotrophins. This pathway, which is independent of the pituitary gland, is activated by an i.c.v. injection of either the stress-related peptide corticotrophin-releasing factor (CRF) or of beta-adrenoceptor agonists, both of which alter androgen release and decrease levels of the peripheral-type benzodiazepine receptor and the steroidogenic acute regulatory protein within Leydig cells. Our original studies used the retrograde transganglionic tracer pseudorabies virus (PRV) to map progression of the virus from the testes to upper brain levels. The present study aimed to extend this work by identifying the regions where CRF and catecholamine neurones represented components of the stress-activated, brain-testicular pathway that prevents testosterone increases. To this end, anaesthetised adult male rats received an intra-testicular injection of PRV. Using immunofluorescence, we identified co-labelling of PRV and either CRF or tyrosine hydroxylase (TH), the enzyme responsible for biogenic amine synthesis. Co-labelling of PRV and CRF was found in the bed nucleus of the stria terminalis, the paraventricular nucleus of the hypothalamus (PVN) and the central amygdala. Co-labelling of PRV and TH was found in the PVN, substantia nigra, A7/Kölliker-Fuse area, area of A5, locus coeruleus, nucleus of solitary tract, area of C3, area of C2 and the area of C1/A1. These results indicate that most cell groups of the ventral noradrenergic pathway have neurones that are a part of the brain-testicular pathway. This suggests that the stress hormones CRF and catecholamines may act as neurotransmitters that signal the pathway to inhibit increases in plasma testosterone levels.

  20. Increased protein expression of LHCG receptor and 17a-hydroxylase/17,20-lyase in human polycystic ovaries

    NARCIS (Netherlands)

    Comim, F.V.; Teerds, K.J.; Hardy, K.; Franks, S.

    2013-01-01

    STUDY QUESTION Does the expression of LHCG receptor (LHCGR) protein and key enzymes in the androgen biosynthetic pathway differ in normal human versus polycystic ovarian tissue? SUMMARY ANSWER LHCGR and 17a-hydroxylase/17-20-lyase (CYP17A1) protein levels are increased in polycystic ovaries (PCOs).

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

    Science.gov (United States)

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

    2015-12-01

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

  2. 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

  3. Roles of the tyrosine isomers meta-tyrosine and ortho-tyrosine in oxidative stress.

    Science.gov (United States)

    Ipson, Brett R; Fisher, Alfred L

    2016-05-01

    The damage to cellular components by reactive oxygen species, termed oxidative stress, both increases with age and likely contributes to age-related diseases including Alzheimer's disease, atherosclerosis, diabetes, and cataract formation. In the setting of oxidative stress, hydroxyl radicals can oxidize the benzyl ring of the amino acid phenylalanine, which then produces the abnormal tyrosine isomers meta-tyrosine or ortho-tyrosine. While elevations in m-tyrosine and o-tyrosine concentrations have been used as a biological marker of oxidative stress, there is emerging evidence from bacterial, plant, and mammalian studies demonstrating that these isomers, particularly m-tyrosine, directly produce adverse effects to cells and tissues. These new findings suggest that the abnormal tyrosine isomers could in fact represent mediators of the effects of oxidative stress. Consequently the accumulation of m- and o-tyrosine may disrupt cellular homeostasis and contribute to disease pathogenesis, and as result, effective defenses against oxidative stress can encompass not only the elimination of reactive oxygen species but also the metabolism and ultimately the removal of the abnormal tyrosine isomers from the cellular amino acid pool. Future research in this area is needed to clarify the biologic mechanisms by which the tyrosine isomers damage cells and disrupt the function of tissues and organs and to identify the metabolic pathways involved in removing the accumulated isomers after exposure to oxidative stress. Published by Elsevier B.V.

  4. Co-localization patterns of neurotensin receptor 1 and tyrosine hydroxylase in brain regions involved in motivation and social behavior in male European starlings.

    Science.gov (United States)

    Merullo, Devin P; Spool, Jeremy A; Zhao, Changjiu; Riters, Lauren V

    2018-04-01

    Animals communicate in distinct social contexts to convey information specific to those contexts, such as sexual or agonistic motivation. In seasonally-breeding male songbirds, seasonal changes in day length and increases in testosterone stimulate sexually-motivated song directed at females for courtship and reproduction. Dopamine and testosterone may act in the same brain regions to stimulate sexually-motivated singing. The neuropeptide neurotensin, acting at the neurotensin receptor 1 (NTR1), can strongly influence dopamine transmission. The goal of this study was to gain insight into the degree to which seasonal changes in physiology modify interactions between neurotensin and dopamine to adjust context-appropriate communication. Male European starlings were examined in physiological conditions that stimulate season-typical forms of communication: late summer/early fall non-breeding condition (low testosterone; birds sing infrequently), late fall non-breeding condition (low testosterone; birds produce non-sexually motivated song), and spring breeding condition (high testosterone; males produce sexually-motivated song). Double fluorescent immunolabeling was performed to detect co-localization patterns between tyrosine hydroxylase (TH; the rate-limiting enzyme in dopamine synthesis) and NTR1 in brain regions implicated in motivation and song production (the ventral tegmental area, medial preoptic nucleus, periaqueductal gray, and lateral septum). Co-localization between TH and NTR1 was present in the ventral tegmental area for all physiological conditions, and the number of co-localized cells did not differ across conditions. Immunolabeling for TH and NTR1 was also present in the other examined regions, although no co-localization was seen. These results support the hypothesis that interactions between NTR1 and dopamine in the ventral tegmental area may modulate vocalizations, but suggest that testosterone- or photoperiod-induced changes in NTR1/TH co

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

    OpenAIRE

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

    1989-01-01

    The impact of experimentally induced diabetes on the expression of rat liver phenylalanine hydroxylase has been investigated. A significant elevation in maximal enzymic activity was observed in diabetes. This was associated with significant increases in the amount of enzyme, the phenylalanine hydroxylase-specific translational activity of hepatic RNA and the abundance of phenylalanine hydroxylase-specific mRNA. These changes in phenylalanine hydroxylase expression were not observed when diabe...

  6. Organisation and tyrosine hydroxylase and calretinin immunoreactivity in the main olfactory bulb of paca (Cuniculus paca): a large caviomorph rodent.

    Science.gov (United States)

    Sasahara, Tais Harumi de Castro; Leal, Leonardo Martins; Spillantini, Maria Grazia; Machado, Márcia Rita Fernandes

    2015-04-01

    The majority of neuroanatomical and chemical studies of the olfactory bulb have been performed in small rodents, such as rats and mice. Thus, this study aimed to describe the organisation and the chemical neuroanatomy of the main olfactory bulb (MOB) in paca, a large rodent belonging to the Hystricomorpha suborder and Caviomorpha infraorder. For this purpose, histological and immunohistochemical procedures were used to characterise the tyrosine hydroxylase (TH) and calretinin (CR) neuronal populations and their distribution. The paca MOB has eight layers: the olfactory nerve layer (ONL), the glomerular layer (GL), the external plexiform layer (EPL; subdivided into the inner and outer sublayers), the mitral cell layer (MCL), the internal plexiform layer (IPL), the granule cell layer (GCL), the periventricular layer and the ependymal layer. TH-ir neurons were found mostly in the GL, and moderate numbers of TH-ir neurons were scattered in the EPL. Numerous varicose fibres were distributed in the IPL and in the GCL. CR-ir neurons concentrated in the GL, around the base of the olfactory glomeruli. Most of the CR-ir neurons were located in the MCL, IPL and GCL. Some of the granule cells had an apical dendrite with a growth cone. The CR immunoreactivity was also observed in the ONL with olfactory nerves strongly immunostained. This study has shown that the MOB organisation in paca is consistent with the description in other mammals. The characterisation and distribution of the population of TH and CR in the MOB is not exclusively to this species. This large rodent shares common patterns to other caviomorph rodent, as guinea pig, and to the myomorph rodents, as mice, rats and hamsters.

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

    International Nuclear Information System (INIS)

    Hei, Ming-Yan; Luo, Ya-Li; Zhang, Xiao-Chun; Liu, Hong; Gao, Ru; Wu, Jing-Jiang

    2011-01-01

    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 2 /92% N 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

  8. 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.

  9. Hemin-Graphene Derivatives with Increased Peroxidase Activities Restrain Protein Tyrosine Nitration.

    Science.gov (United States)

    Xu, Huan; Yang, Zhen; Li, Hailing; Gao, Zhonghong

    2017-12-14

    Protein tyrosine nitration is implicated in the occurrence and progression of pathological conditions involving free radical reactions. It is well recognized that hemin can catalyze protein tyrosine nitration in the presence of nitrite and hydrogen peroxide. Generally, the catalytic efficiency is positively correlated to its peroxidase activity. In this study, however, it is found that the efficiency of hemin in catalyzing protein tyrosine nitration is largely suppressed after functionalization with graphene derivatives, even though its peroxidase-like activity is more than quadrupled. Further studies show that the oxidation of tyrosine is still observed for these composites; dityrosine formation, however, is greatly inhibited. Furthermore, these composites also exhibit strong effects on the oxidation of nitrite into nitrate. Therefore, we propose a mechanism in which hemin-graphene derivatives facilitate the oxidation of tyrosine and nitrite to produce tyrosyl radicals and nitrogen dioxide radicals in the presence of hydrogen peroxide, but graphene interlayers serve as barriers that hinder radical-radical coupling reactions; consequently, protein tyrosine nitration is restrained. This property of hemin-graphene derivatives, by which they catalyze substrate oxidation but suppress radical-radical coupling reactions, shows their great potential in selective oxidation procedures for byproduct removal. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

    Spasojevic, Natasa; Jovanovic, Predrag; Dronjak, Sladjana

    2015-03-01

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

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

    Directory of Open Access Journals (Sweden)

    NATASA SPASOJEVIC

    2015-03-01

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

  12. Genetics Home Reference: 21-hydroxylase deficiency

    Science.gov (United States)

    ... adrenal hyperplasias that impair hormone production and disrupt sexual development. 21-hydroxylase deficiency is responsible for about 95 ... excess production of androgens leads to abnormalities of sexual development in people with 21-hydroxylase deficiency . A lack ...

  13. 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

  14. 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...... and used in treatment. A process for preparing the crystalline form of human dopamine β-hydroxylase is also provided....

  15. Increasing production yield of tyrosine and mevalonate through inhibition of biomass formation

    DEFF Research Database (Denmark)

    Li, Songyuan; Jendresen, Christian Bille; Nielsen, Alex Toftgaard

    2016-01-01

    , in particular, resulted in an increase in mass yield of mevalonate and tyrosine by 80% and 50%, respectively. By tracking production and biomass concentrations, it was observed that the production was maintained for more than 10 h after inhibition of cell growth, despite cell maintenance requirements...

  16. Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

    DEFF Research Database (Denmark)

    Jacob, K K; Sap, J; Stanley, F M

    1998-01-01

    A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin...... is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due...... to reduced phosphorylation or kinase activity of the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not mitogen-activated protein kinase dependent...

  17. Heterogeneous expression of cholesterol 7α-hydroxylase and sterol 27- hydroxylase genes in the rat liver lobulus

    NARCIS (Netherlands)

    Twisk, J.; Hoekman, M.F.M.; Mager, W.H.; Moorman, A.F.M.; Boer, P.A.J. de; Scheja, L.; Princen, H.M.G.; Gebhardt, R.

    1995-01-01

    We investigated the lobular localization and molecular level of expression of cholesterol 7α-hydroxylase and sterol 27-hydroxylase, two key enzymes in bile acid synthesis, in isolated periportal and pericentral hepatocytes and by in situ hybridization of rat liver. Enzyme activity, mRNA, and gene

  18. 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

  19. Inhibition of biofilm formation by D-tyrosine: Effect of bacterial type and D-tyrosine concentration.

    Science.gov (United States)

    Yu, Cong; Li, Xuening; Zhang, Nan; Wen, Donghui; Liu, Charles; Li, Qilin

    2016-04-01

    D-Tyrosine inhibits formation and triggers disassembly of bacterial biofilm and has been proposed for biofouling control applications. This study probes the impact of D-tyrosine in different biofilm formation stages in both G+ and G- bacteria, and reveals a non-monotonic correlation between D-tyrosine concentration and biofilm inhibition effect. In the attachment stage, cell adhesion was studied in a flow chamber, where D-tyrosine caused significant reduction in cell attachment. Biofilms formed by Pseudomonas aeruginosa and Bacillus subtilis were characterized by confocal laser scanning microscopy as well as quantitative analysis of cellular biomass and extracellular polymeric substances. D-Tyrosine exhibited strong inhibitive effects on both biofilms with an effective concentration as low as 5 nM; the biofilms responded to D-tyrosine concentration change in a non-monotonic, bi-modal pattern. In addition, D-tyrosine showed notable and different impact on EPS production by G+ and G- bacteria. Extracellular protein was decreased in P. aeruginosa biofilms, but increased in those of B. subtilis. Exopolysaccharides production by P. aeruginosa was increased at low concentrations and reduced at high concentrations while no impact was found in B. subtilis. These results suggest that distinct mechanisms are at play at different D-tyrosine concentrations and they may be species specific. Dosage of D-tyrosine must be carefully controlled for biofouling control applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Phenylalanine metabolism in isolated rat liver cells. Effects of glucagon and diabetes.

    OpenAIRE

    Carr, F P; Pogson, C I

    1981-01-01

    1. Methods are described for monitoring the metabolic flux through phenylalanine hydroxylase, the tyrosine catabolic pathway and phenylalanine: pyruvate transaminase in isolated liver cell incubations. 2. The relationship between hydroxylase flux and phenylalanine concentration is sigmoidal. 3. Glucagon increases hydroxylase activity at low, near-physiological, substrate concentrations only. The hormone does not affect the rate of formation of phenylpyruvate. 4. Experimental diabetes (for 10 ...

  1. Development of vitamin D3 25-hydroxylase activity in rat liver microsomes

    International Nuclear Information System (INIS)

    Thierry-Palmer, M.; Cullins, S.; Rashada, S.; Gray, T.K.; Free, A.

    1986-01-01

    The authors have determined the ontogeny of vitamin D 3 25-hydroxylase activity in rat liver microsomes. Microsomes from fetuses, neonates, and their mothers were incubated with 44 nM 3 H-vitamin D 3 in the presence of an NADPH generating system, oxygen, KCl, and MgCl 2 . Lipid extracts of the incubation samples were partially purified by thin-layer chromatography. Tritiated 25-hydroxy vitamin D 3 (250HD 3 ) was analyzed by high-pressure liquid chromatography using 94/6 hexane/isopropanol. Production rate for 250HD 3 in the mothers ranged from 0.22 to 0.30 pmol/mg protein/hr. Activities in the fetuses and neonates were 2.1, 12.9, 32.0, 35.8, and 71.0% of that of their mothers at -3, 0, 2, 7, and 15 days of age. The cytosolic fraction protected the substrate from degradation, stimulated the vitamin D 3 25-hydroxylase reaction in neonates and mothers (1.4 to 1.7 fold increase), and was absolutely required for 25-hydroxylase activity in fetuses. These data suggest that microsomal vitamin D 3 25-hydroxylase activity develops slowly and approaches full activity near the weaning stage. A cytosolic factor present as early as -3 days of age stimulates the activity of the microsomal vitamin D 3 25-hydroxylase

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    W. H. Davin Townley-Tilson

    2015-01-01

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

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

    Science.gov (United States)

    Rastogi, R B; Singhal, R L

    1976-09-01

    In neonatal rats, administration of l-triiodothyronine (10 mug/100 g/day) for 30 days presented signs of hyperthyroidism which included accelerated development of a variety of physical and behavioral characteristics accompanying maturation. The spontaneous motor activity was increased by 69%. Exposure of developing rats to thyroid hormone significantly increased the endogenous concentration of striatal tyrosine and the activity of tyrosine hydroxylase as well as the levels of dopamine in several brain regions. The concentration of striatal homovanillic acid and 3,4-dihydroxyphenylacetic acid, the chief metabolites of dopamine, was also increased and the magnitude of change was greater than the rise in dopamine. Despite increases in the activity of tyrosine hydroxylase and the availability of the substrate tyrosine, the steady-state levels of norepinephrine remained unaltered in various regions of brain except in cerebellum. Futhermore, neonatal hyperthyroidism significantly increased the levels of midbrain tryptophan and tryptophan hydroxylase activity but produced no change in 5-hydroxytryptamine levels of several discrete brain regions, except hypothalamus and cerebellum where its concentration was slightly decreased. However, the 5-hydroxyindoleacetic acid levels were enhanced in hypothalamus, ponsmedulla, midbrain, striatum and hippocampus. The elevated levels of 5-hydroxyindoleacetic acid did not seem to be due to increased intraneuronal deamination of 5-hydroxytryptamine since monoamine oxidase activity was not affected in cerebral cortex and midbrain of hyperthyroid rats. The data demonstrate that hyperthyroidism significantly increased the synthesis as well as the utilization of catecholamines and 5-hydroxytryptamine in maturing brain. Since the mature brain is known to respond differently to thyroid hormone action than does the developing brain, the effect of L-triiodothyronine treatment on various putative neurohumors also was examined in adult rats

  5. 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

  6. Formation of tyrosine isomers in aqueous phenylalanine solutions by gamma irradiation

    International Nuclear Information System (INIS)

    Aflaki, F.; Salahinejad, M.; Roozbehani, A.

    2009-01-01

    Ortho-tyrosine detection method can be used for detection of irradiated protein rich foods. Tyrosine isomers produced by gamma radiation of aqueous phenylalanine solutions at wide dose levels (0.1-50 k Gy) were examined to obtain basic information for o-tyrosine detection method of irradiated foods. Determination of tyrosines produced in aqueous phenylalanine solutions were carried out by high performance liquid chromatography and fluorescence detection. The detection limit of o-tyrosine was 0.01 ppm and the linear range of calibration and the relative standard deviation of analysis was 50 ng and 4-13%, respectively. The amounts of the tyrosines increased with the irradiation level up to 10 k Gy and no further tyrosine formation was observed when the dose level was increased. At a constant dose level, the yield of tyrosines initially increased with the phenylalanine concentration, while with further increase of phenylalanine concentration no effect on increase of tyrosine yield was observed. When the dose rate was varying from 2.3 k Gy/h to 1.2 k Gy/h with a total amount of 10 k Gy in each case, there was no significant effect on tyrosine isomers formation was observed. Also the results showed that tyrosine yield was affected by temperature, p H and the presence of oxygen

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

    Science.gov (United States)

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

    1991-02-01

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

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

    Science.gov (United States)

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

    2014-04-01

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

  9. Electrode Potentials of l-Tryptophan, l-Tyrosine, 3-Nitro-l-tyrosine, 2,3-Difluoro-l-tyrosine, and 2,3,5-Trifluoro-l-tyrosine.

    Science.gov (United States)

    Mahmoudi, Leila; Kissner, Reinhard; Nauser, Thomas; Koppenol, Willem H

    2016-05-24

    Electrode potentials for aromatic amino acid radical/amino acid couples were deduced from cyclic voltammograms and pulse radiolysis experiments. The amino acids investigated were l-tryptophan, l-tyrosine, N-acetyl-l-tyrosine methyl ester, N-acetyl-3-nitro-l-tyrosine ethyl ester, N-acetyl-2,3-difluoro-l-tyrosine methyl ester, and N-acetyl-2,3,5-trifluoro-l-tyrosine methyl ester. Conditional potentials were determined at pH 7.4 for all compounds listed; furthermore, Pourbaix diagrams for l-tryptophan, l-tyrosine, and N-acetyl-3-nitro-l-tyrosine ethyl ester were obtained. Electron transfer accompanied by proton transfer is reversible, as confirmed by detailed analysis of the current waves, and because the slopes of the Pourbaix diagrams obey Nernst's law. E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH) at pH 7 are 0.99 ± 0.01 and 0.97 ± 0.01 V, respectively. Pulse radiolysis studies of two dipeptides that contain both amino acids indicate a difference in E°' of approximately 0.06 V. Thus, in small peptides, we recommend values of 1.00 and 0.96 V for E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH), respectively. The electrode potential of N-acetyl-3-nitro-l-tyrosine ethyl ester is higher, while because of mesomeric stabilization of the radical, those of N-acetyl-2,3-difluoro-l-tyrosine methyl ester and N-acetyl-2,3,5-trifluoro-l-tyrosine methyl ester are lower than that of tyrosine. Given that the electrode potentials at pH 7 of E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH) are nearly equal, they would be, in principle, interchangeable. Proton-coupled electron transfer pathways in proteins that use TrpH and TyrOH are thus nearly thermoneutral.

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

    Science.gov (United States)

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

    2016-06-29

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

  11. 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.

  12. 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.

  13. Leptin Intake at Physiological Doses Throughout Lactation in Male Wistar Rats Normalizes the Decreased Density of Tyrosine Hydroxylase-Immunoreactive Fibers in the Stomach Caused by Mild Gestational Calorie Restriction

    Directory of Open Access Journals (Sweden)

    Nara Szostaczuk

    2018-03-01

    Full Text Available Introduction: Gestational under nutrition in rats has been shown to decrease expression of sympathetic innervation markers in peripheral tissues of offspring, including the stomach. This has been linked to lower gastric secretion and decreased circulating levels of ghrelin. Considering the critical role of leptin intake during lactation in preventing obesity and reversing adverse developmental programming effects, we aimed to find out whether leptin supplementation may reverse the above mentioned alterations caused by mild gestational calorie restriction.Methods: Three groups of male rats were studied at a juvenile age (25 days old and during adulthood (3 and 6 months old: the offspring of ad libitum fed dams (controls, the offspring of dams that were diet restricted (20% from days 1 to 12 of gestation (CR, and CR rats supplemented with a daily oral dose of leptin (equivalent to 5 times the average amount they could receive each day from maternal milk throughout lactation (CR-Leptin. The density of TyrOH-immunoreactive (TyrOH+ fibers and the levels of Tyrosine hydroxylase (TyrOH—used as potential markers of functional sympathetic innervation—were measured in stomach. Plasma leptin and ghrelin levels were also determined.Results: Twenty five-day-old CR rats, but not CR-Leptin rats, displayed lower density of TyrOH+ fibers (−46% and TyrOH levels (−47% in stomach compared to controls. Alterations in CR animals were mitigated at 6 months of age, and differences were not significant. Adult CR-Leptin animals showed higher plasma ghrelin levels than CR animals, particularly at 3 months (+16%, and a lower leptin/ghrelin ratio (−28 and −37% at 3 and 6 months, respectively.Conclusion: Leptin intake during lactation is able to reverse the alterations in the density of TyrOH+ fibers in the stomach and normalize the increased leptin/ghrelin ratio linked to a mild gestational calorie restriction in rats, supporting the relevance of leptin as an

  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. A sandwich immunoassay for human prolyl 4-hydroxylase using monoclonal antibody

    International Nuclear Information System (INIS)

    Yoshida, Shinichi

    1986-01-01

    Monoclonal antibody was used in a sandwich enzyme immunoassay and in a radioimmunoassay for human serum immunoreactive prolyl 4-hydroxylase. The enzyme immunoassay utilized a monoclonal antibody as a solid phase and horseradish peroxidase-labeled rabbit antibody to human prolyl 4-hydroxylase as a conjugate. Sensitivity was 0.1 ng of enzyme per tube. With a conjugate purified by an enzyme-bound affinity column, sensitivity was increased to 0.01 ng per tube, and linearity was obtained between 0.01 to 30 ng per tube. The radioimmunoassay used a 125 I-labeled rabbit antibody (IgG) as the conjugate. Sensitivity of this technique was 0.4 ng of enzyme per tube. (Auth.)

  16. 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

  17. Conversion of p-tyrosine to p-tyramine in the isolated perfused rat kidney: Modulation by perfusate concentrations of p-tyrosine

    International Nuclear Information System (INIS)

    Brier, M.E.; Bowsher, R.R.; Henry, D.P.; Mayer, P.R.

    1991-01-01

    The authors used the isolated perfused rat kidney to evaluate the role of renal decarboxylation of p-tyrosine as the source of urinary p-tyramine. Kidneys were perfused with concentrations of p-tyrosine ranging from 0.02 mM to 2.0 mM. p-Tyramine was measured by a sensitive and specific radioenzymatic assay. An increase in the perfusate concentration of p-tyrosine resulted in a significant increase in p-tyramine production that was blocked by the addition of NSD-1015, an inhibitor of aromatic-1-amino decarboxylase (AADC). They conclude p-tyrosine is the precursor for the renal production of p-tyramine, renal AADC catalyzes the formation of urinary p-tyramine, synthesized p-tyramine is predominantly excreted in the urine, and p-tyramine synthesis is modulated by the arterial delivery of p-tyrosine to the kidney

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

    Science.gov (United States)

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

    2001-01-01

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

  19. 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.

  20. 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

  1. Homeostatic mechanisms in dopamine synthesis and release: a mathematical model

    Directory of Open Access Journals (Sweden)

    Nijhout H Frederik

    2009-09-01

    Full Text Available Abstract Background Dopamine is a catecholamine that is used as a neurotransmitter both in the periphery and in the central nervous system. Dysfunction in various dopaminergic systems is known to be associated with various disorders, including schizophrenia, Parkinson's disease, and Tourette's syndrome. Furthermore, microdialysis studies have shown that addictive drugs increase extracellular dopamine and brain imaging has shown a correlation between euphoria and psycho-stimulant-induced increases in extracellular dopamine 1. These consequences of dopamine dysfunction indicate the importance of maintaining dopamine functionality through homeostatic mechanisms that have been attributed to the delicate balance between synthesis, storage, release, metabolism, and reuptake. Methods We construct a mathematical model of dopamine synthesis, release, and reuptake and use it to study homeostasis in single dopaminergic neuron terminals. We investigate the substrate inhibition of tyrosine hydroxylase by tyrosine, the consequences of the rapid uptake of extracellular dopamine by the dopamine transporters, and the effects of the autoreceoptors on dopaminergic function. The main focus is to understand the regulation and control of synthesis and release and to explicate and interpret experimental findings. Results We show that the substrate inhibition of tyrosine hydroxylase by tyrosine stabilizes cytosolic and vesicular dopamine against changes in tyrosine availability due to meals. We find that the autoreceptors dampen the fluctuations in extracellular dopamine caused by changes in tyrosine hydroxylase expression and changes in the rate of firing. We show that short bursts of action potentials create significant dopamine signals against the background of tonic firing. We explain the observed time courses of extracellular dopamine responses to stimulation in wild type mice and mice that have genetically altered dopamine transporter densities and the observed

  2. Amino acids in health and disease: New perspectives

    Energy Technology Data Exchange (ETDEWEB)

    Kaufman, S.

    1987-01-01

    This book contains 33 selections. Some of the titles are: Regulation of Adrenal Tyrosine Hydroxylase Gene Expression During Cold Stress; The Molecular Genetics of Phenylketonuria; Prospects for Somatic Gene Therapy of Phenylketonuria; Behavioral Effects of Sugar; Effects of Tyrosine and Tryptophan on Blood Pressure in the Rat; and The Enzymology of the Aromatic Amino Acid Hydroxylases.

  3. 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.

  4. 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.

  5. 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

  6. Radiolytic dimerization of tyrosine in alkaline solutions of poly-L-tyrosine, glycyl-L-tyrosine and tyrosine

    International Nuclear Information System (INIS)

    Boguta, G.; Dancewicz, A.M.

    1982-01-01

    Blue fluorescence characteristic of dityrosine appeared in γ-irradiated solutions of tyrosine, glycyl-L-tyrosine or polytyrosine (MW 110,000). The intensity of fluorescence was used for the determination of the dityrosine concentration in hydrolysed samples. The radiation-induced formation of dityrosine depended on pH and on the presence of oxygen during radiolysis carried out with a total dose of the order of 1000 Gy. The presence of oxygen in the system suppressed the formation of dityrosine in solution at low or neutral pH but had no effect on this process in alkaline solutions. Except for the radiolysis of air-saturated poly-L-tyrosine solutions, where G(Dityrosine) = 0.35, the yields of dityrosine at high pH were lower than the yields obtained during radiolysis at low pH and in the absence of oxygen. (author)

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

    Science.gov (United States)

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

    1985-08-01

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

  8. Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus

    International Nuclear Information System (INIS)

    Nakayama, Yuji; Kawana, Akiko; Igarashi, Asae; Yamaguchi, Naoto

    2006-01-01

    Chk tyrosine kinase phosphorylates Src-family kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. In this study, we explored the role of the N-terminal unique domain of Chk in nuclear localization and Chk-induced tyrosine phosphorylation in the nucleus. In situ binding experiments showed that the N-terminal domain of Chk was associated with the nucleus and the nuclear matrix. The presence of the N-terminal domain of Chk led to a fourfold increase in cell population exhibiting Chk-induced tyrosine phosphorylation in the nucleus. Expression of Chk but not kinase-deficient Chk induced tyrosine phosphorylation of a variety of proteins ranging from 23 kDa to ∼200 kDa, especially in Triton X-100-insoluble fraction that included chromatin and the nuclear matrix. Intriguingly, in situ subnuclear fractionations revealed that Chk induced tyrosine phosphorylation of proteins that were associated with the nuclear matrix. These results suggest that various unidentified substrates of Chk, besides Src-family kinases, may be present in the nucleus. Thus, our findings indicate that the importance of the N-terminal domain to Chk-induced tyrosine phosphorylation in the nucleus, implicating that these nuclear tyrosine-phosphorylated proteins may contribute to inhibition of cell proliferation

  9. Tyrosine transport in winter flounder intestine: Interaction with Na+-K+-2Cl- cotransport

    International Nuclear Information System (INIS)

    Musch, M.W.; McConnell, F.M.; Goldstein, L.; Field, M.

    1987-01-01

    Tyrosine absorption across the brush border of the intestinal epithelium of the winter flounder Pseudopleuronectes americanus was studied in Ussing chambers modified to determine early rates of uptake. At 0.1 mM tyrosine, the 4-min rate of uptake (influx) of tyrosine across the brush border averaged 37.5 nmol·cm -2 ·h -1 . Omission of Na decreased influx by 60%, indicting that tyrosine influx occurs, at least in part, by a Na-coupled process. Ouabain inhibited influx by 80%. Inhibition of brush border Na + -K + -2Cl - cotransport by bumetanide, 8-bromo-cyclic GMP, or Cl replacement stimulated tyrosine influx 2.5- to 4-fold. However, atriopeptin III, which also inhibits Na + -K + -2Cl - cotransport, did not stimulate tyrosine influx. Cyclic AMP, which does not appear to inhibit ion cotransport, did not stimulate tyrosine influx. Both cyclic GMP and bumetanide also stimulated the net mucosa-to-serosa tyrosine flux (43 and 29%, respectively) and increased the cellular concentration of tyrosine by 50%. Thus tyrosine's influx is increased to a greater extent than is its transmural flux or its cellular concentration, suggesting that the main change occurs at the brush border and represents large increases in both influx and efflux of tyrosine across this membrane

  10. Therapeutic Potential of a Prolyl Hydroxylase Inhibitor FG-4592 for Parkinson’s Diseases in Vitro and in Vivo: Regulation of Redox Biology and Mitochondrial Function

    Directory of Open Access Journals (Sweden)

    Xuan Li

    2018-04-01

    Full Text Available As the main transcription factor that regulates the cellular responses to hypoxia, Hypoxia-inducible factor-1α (HIF-1α plays an important role in the pathogenesis of Parkinson’s disease (PD. HIF-1α is normally degraded through ubiquitination after hydroxylation by prolyl hydroxylases (PHD. Emerging evidence has suggested that HIF PHD inhibitors (HIF-PHI may have neuroprotective effects on PD through increasing HIF-1α levels. However, the therapeutic benefit of HIF-PHI for PD remains poorly explored due to the lack of proper clinical compounds and understanding of the underlying molecular mechanisms. In this study, we examined the therapeutic benefit of a new HIF-PHI, FG-4592, which is currently in phase 3 clinical trials to treat anemia in patients with chronic kidney diseases (CKD in PD models. FG-4592 attenuates MPP+ -induced apoptosis and loss of tyrosine hydroxylase (TH in SH-SY5Y cells. Pretreatment with FG-4592 mitigates MPP+-induced loss of mitochondrial membrane potential (MMP, mitochondrial oxygen consumption rate (OCR, production of reactive oxygen species (ROS and ATP. Furthermore, FG-4592 counterbalances the oxidative stress through up-regulating nuclear factor erythroid 2 p45-related factor 2 (Nrf-2, heme oxygenase-1 (HO-1 and superoxide dismutase 2 (SOD2. FG-4592 treatment also induces the expression of Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α through increasing the phosphorylation of AMP-activated protein kinase (AMPK. In MPTP-treated mice, FG-4592 protects against MPTP-induced loss of TH-positive neurons of substantia nigra and attenuates behavioral impairments. Collectively, our study demonstrates that FG-4592 is a promising therapeutic strategy for PD through improving the mitochondrial function under oxidative stress.

  11. 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

  12. Eating to stop: Tyrosine supplementation enhances inhibitory control but not response execution

    NARCIS (Netherlands)

    Colzato, L.S.; Jongkees, B.J.; Sellaro, R.; van den Wildenberg, W.P.M.; Hommel, B.

    2014-01-01

    Animal studies and research in humans have shown that the supplementation of tyrosine, or tyrosine-containing diets, increase the plasma tyrosine and enhance brain dopamine (DA). However, the strategy of administering tyrosine (and the role of DA therein) to enhance cognition is unclear and heavily

  13. The Tyrosine Aminomutase TAM1 Is Required for β-Tyrosine Biosynthesis in Rice

    Science.gov (United States)

    Yan, Jian; Aboshi, Takako; Teraishi, Masayoshi; Strickler, Susan R.; Spindel, Jennifer E.; Tung, Chih-Wei; Takata, Ryo; Matsumoto, Fuka; Maesaka, Yoshihiro; McCouch, Susan R.; Okumoto, Yutaka; Mori, Naoki; Jander, Georg

    2015-01-01

    Non-protein amino acids, often isomers of the standard 20 protein amino acids, have defense-related functions in many plant species. A targeted search for jasmonate-induced metabolites in cultivated rice (Oryza sativa) identified (R)-β-tyrosine, an isomer of the common amino acid (S)-α-tyrosine in the seeds, leaves, roots, and root exudates of the Nipponbare cultivar. Assays with 119 diverse cultivars showed a distinct presence/absence polymorphism, with β-tyrosine being most prevalent in temperate japonica cultivars. Genetic mapping identified a candidate gene on chromosome 12, which was confirmed to encode a tyrosine aminomutase (TAM1) by transient expression in Nicotiana benthamiana and in vitro enzyme assays. A point mutation in TAM1 eliminated β-tyrosine production in Nipponbare. Rice cultivars that do not produce β-tyrosine have a chromosome 12 deletion that encompasses TAM1. Although β-tyrosine accumulation was induced by the plant defense signaling molecule jasmonic acid, bioassays with hemipteran and lepidopteran herbivores showed no negative effects at physiologically relevant β-tyrosine concentrations. In contrast, root growth of Arabidopsis thaliana and other tested dicot plants was inhibited by concentrations as low as 1 μM. As β-tyrosine is exuded into hydroponic medium at higher concentrations, it may contribute to the allelopathic potential of rice. PMID:25901084

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

    International Nuclear Information System (INIS)

    Wilson, S.P.

    1991-01-01

    Insulin-like growth factor I (IGF-I) increased both the contents of proenkephalin derived enkephalin-containing peptides and the activity of dopamine β-hydroxylase in bovine adrenal chromaffin cells. These increases in dopamine β-hydroxylase and enkephalin-containing peptides continued for at least 8 days. The half-maximal IGF-I concentration for these effects was ∼ 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 [ 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

  15. 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.

  16. 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.

  17. Behavioral and cognitive effects of tyrosine intake in healthy human adults

    NARCIS (Netherlands)

    Hase, Adrian; Jung, Sophie E.; aan het Rot, Marije

    2015-01-01

    The amino acid tyrosine is the precursor to the catecholamine neurotransmitters dopamine and norepinephrine. Increasing tyrosine uptake may positively influence catecholamine-related psychological functioning. We conducted a systematic review to examine the effects of tyrosine on behavior and

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

    Science.gov (United States)

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

    2013-01-01

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

  19. AAV-mediated gene transfer of the obesity-associated gene Etv5 in rat midbrain does not affect energy balance or motivated behavior.

    Directory of Open Access Journals (Sweden)

    Arjen J Boender

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

  20. 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...... residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signalling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation...

  1. 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...

  2. Genetics Home Reference: dopamine beta-hydroxylase deficiency

    Science.gov (United States)

    ... common features include an unusually large range of joint movement (hypermobility) and muscle weakness. Related Information What ... Dopamine beta-hydroxylase deficiency Washington Univeristy, St. Louis: Neuromuscular Disease Center Patient Support and Advocacy Resources (1 ...

  3. Evaluation of o-[11C]methyl-L-tyrosine and o-[18F]fluoromethyl-L-tyrosine as tumor imaging tracers by PET

    International Nuclear Information System (INIS)

    Ishiwata, Kiichi; Kawamura, Kazunori; Wang Weifang; Furumoto, Shozo; Kubota, Kazuo; Pascali, Claudio; Bogni, Anna; Iwata, Ren

    2004-01-01

    We investigated the potential of O-[ 11 C]methyl-L-tyrosine and O-[ 18 F]fluoromethyl-L-tyrosine as positron-emitting tracers for tumor imaging. The two tracers had similar distribution patterns in rats bearing AH109A hepatoma, with pancreas and, on a lesser extent, AH109A showing the highest uptake. Uptake of both tracers in the AH109A and uptake ratios of AH109A-to-tissues (with the exception of AH109A-to-bone) gradually increased for 60 min. O-[ 11 C]methyl-L-tyrosine was metabolically stable, whereas a negligible low amount of metabolites was observed for O-[ 18 F]fluoromethyl-L-tyrosine. Both tracers showed the potential for tumor imaging

  4. Increased dependence of leucine in posttraumatic sepsis: leucine/tyrosine clearance ratio as an indicator of hepatic impairment in septic multiple organ failure syndrome.

    Science.gov (United States)

    Pittiruti, M; Siegel, J H; Sganga, G; Coleman, B; Wiles, C E; Belzberg, H; Wedel, S; Placko, R

    1985-09-01

    The body clearance of 10 plasma amino acids (AA) was determined from the rate of compared muscle-released AA and AA administered by infusion of total parenteral nutrition (TPN) compared to their estimated extracellular (ECW) pool in patients with multiple trauma with (n = 10) or without (n = 16) sepsis at 8-hour intervals. In both nonseptic and septic trauma, increasing TPN increased the mean clearance rate of all infused AA. When the individual AA clearance rates were normalized by the total AA infusion rate, regression-covariance analysis revealed that patients with sepsis had relatively impaired clearances of alanine (p less than 0.01) and methionine, proline, phenylalanine, and tyrosine p less than 0.05 for all). In contrast, the clearances of branched-chain AA (BCAA) valine and isoleucine were maintained, and the clearance of leucine was higher (p less than 0.05) in trauma patients with sepsis than in those without. At any AA infusion rate, compared with surviving patients with sepsis (p less than 0.05), patients who developed fatal multiple organ failure syndrome (MOFS) showed increased clearances of all BCAA with further impaired clearance of tyrosine. The clearance ratio of leucine/tyrosine was increased in MOFS at any AA infusion rate (p less than 0.0001), was an indicator of severity, and, if persistent, was a manifestation of a fatal outcome. Because tyrosine metabolism occurs almost entirely in the liver while leucine can be utilized by viscera and muscle, these data suggest early and progressive septic impairment of the pattern of hepatic uptake and oxidation of AA with a greater body dependence on BCAA, especially leucine, as septic MOFS develops.

  5. Functionalization of protected tyrosine via Sonogashira reaction: synthesis of 3-(1,2,3-triazolyl)-tyrosine.

    Science.gov (United States)

    Vasconcelos, Stanley N S; Shamim, Anwar; Ali, Bakhat; de Oliveira, Isadora M; Stefani, Hélio A

    2016-05-01

    1,2,3-Triazol tyrosines were synthesized from tyrosine alkynes that were in turn prepared via Sonogashira cross-coupling reaction. The tyrosine alkynes were subjected to click-chemistry reaction conditions leading to the corresponding 3-(1,2,3-triazolyl)-tyrosines in yields ranging from moderate to good.

  6. 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

    Tryptophan hydroxylase (TPH) is a mononuclear non-heme iron enzyme, which catalyzes the reaction between tryptophan, O2, and tetrahydrobiopterin (BH4) to produce 5-hydroxytryptophan and 4a-hydroxytetrahydrobiopterin. This is the first and rate-limiting step in the biosynthesis of the neurotransmi......Tryptophan hydroxylase (TPH) is a mononuclear non-heme iron enzyme, which catalyzes the reaction between tryptophan, O2, and tetrahydrobiopterin (BH4) to produce 5-hydroxytryptophan and 4a-hydroxytetrahydrobiopterin. This is the first and rate-limiting step in the biosynthesis...... 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...

  7. 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.

  8. Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Macek, B

    2006-01-01

    for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (Sc......SSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically...... by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation...

  9. 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...

  10. 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.

  11. 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.

  12. Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides.

    Science.gov (United States)

    Csernetics, Árpád; Tóth, Eszter; Farkas, Anita; Nagy, Gábor; Bencsik, Ottó; Vágvölgyi, Csaba; Papp, Tamás

    2015-02-01

    Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates β-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of β-carotene (i.e. zeaxanthin and β-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of β-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than β-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and β-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the β-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene.

  13. High frequency of cytolytic 21-hydroxylase-specific CD8+ T cells in autoimmune Addison's disease patients.

    Science.gov (United States)

    Dawoodji, Amina; Chen, Ji-Li; Shepherd, Dawn; Dalin, Frida; Tarlton, Andrea; Alimohammadi, Mohammad; Penna-Martinez, Marissa; Meyer, Gesine; Mitchell, Anna L; Gan, Earn H; Bratland, Eirik; Bensing, Sophie; Husebye, Eystein S; Pearce, Simon H; Badenhoop, Klaus; Kämpe, Olle; Cerundolo, Vincenzo

    2014-09-01

    The mechanisms behind destruction of the adrenal glands in autoimmune Addison's disease remain unclear. Autoantibodies against steroid 21-hydroxylase, an intracellular key enzyme of the adrenal cortex, are found in >90% of patients, but these autoantibodies are not thought to mediate the disease. In this article, we demonstrate highly frequent 21-hydroxylase-specific T cells detectable in 20 patients with Addison's disease. Using overlapping 18-aa peptides spanning the full length of 21-hydroxylase, we identified immunodominant CD8(+) and CD4(+) T cell responses in a large proportion of Addison's patients both ex vivo and after in vitro culture of PBLs ≤20 y after diagnosis. In a large proportion of patients, CD8(+) and CD4(+) 21-hydroxylase-specific T cells were very abundant and detectable in ex vivo assays. HLA class I tetramer-guided isolation of 21-hydroxylase-specific CD8(+) T cells showed their ability to lyse 21-hydroxylase-positive target cells, consistent with a potential mechanism for disease pathogenesis. These data indicate that strong CTL responses to 21-hydroxylase often occur in vivo, and that reactive CTLs have substantial proliferative and cytolytic potential. These results have implications for earlier diagnosis of adrenal failure and ultimately a potential target for therapeutic intervention and induction of immunity against adrenal cortex cancer. Copyright © 2014 by The American Association of Immunologists, Inc.

  14. 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

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

    Science.gov (United States)

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

    1985-10-25

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

  16. 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.

  17. Detection method of prawn irradiated in frozen state using tyrosine isomers as a marker

    International Nuclear Information System (INIS)

    Oikawa, H.; Satomi, M.; Omura, Y.; Yano, Y.

    2001-01-01

    Internationally the use of food irradiation has been expanding. And therefore a method is needed to detect whether food has been irradiated or not. We examined the content of the tyrosine isomers, m-tyrosine and omicron-tyrosine, of prawns irradiated in the frozen state (< -30 deg C) as a marker of the detection method. The tyrosine isomer content linearly increased with increasing dose, and the level of tyrosine isomers in the frozen-irradiated prawn was 50 - 60 % of the un frozen ones. But the difference in the content of tyrosine isomers between non-irradiated and irradiated at 5.0 kGy, that is the approved dose for frozen shellfish in countries where this technique is approved, is enough for discrimination. In addition, the content of tyrosine isomers showed little change during the frozen storage for 120 days. So we think the method using tyrosine isomers is suitable for practical use in Japan for imports of many kinds of frozen shellfish

  18. The mechanism of the tyrosine transporter TyrP supports a proton motive tyrosine decarboxylation pathway in Lactobacillus brevis

    NARCIS (Netherlands)

    Wolken, WAM; Lucas, PM; Lonvaud-Funel, A; Lolkema, JS; Wolken, Wout A.M.; Lucas, Patrick M.

    The tyrosine decarboxylase operon of Lactobacillus brevis IOEB9809 contains, adjacent to the tyrosine decarboxylase gene, a gene for TyrP, a putative tyrosine transporter. The two genes potentially form a proton motive tyrosine decarboxylation pathway. The putative tyrosine transporter gene of L.

  19. 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.

  20. Chlorinated tyrosine derivatives in insect cuticle

    DEFF Research Database (Denmark)

    Andersen, Svend Olav

    2004-01-01

    A method for quantitative measurement of 3-monochlorotyrosine and 3,5-dichlorotyrosine in insect cuticles is described, and it is used for determination of their distribution in various cuticular regions in nymphs and adults of the desert locust, Schistocerca gregaria. The two chlorinated tyrosine......, 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...

  1. Role of Tyrosine Isomers in Acute and Chronic Diseases Leading to Oxidative Stress - A Review.

    Science.gov (United States)

    Molnár, Gergő A; Kun, Szilárd; Sélley, Eszter; Kertész, Melinda; Szélig, Lívia; Csontos, Csaba; Böddi, Katalin; Bogár, Lajos; Miseta, Attila; Wittmann, István

    2016-01-01

    Oxidative stress plays a major role in the pathogenesis of a variety of acute and chronic diseases. Measurement of the oxidative stress-related end products may be performed, e.g. that of structural isomers of the physiological para-tyrosine, namely meta- and ortho-tyrosine, that are oxidized derivatives of phenylalanine. Recent data suggest that in sepsis, serum level of meta-tyrosine increases, which peaks on the 2(nd) and 3(rd) days (ptyrosine excretion correlated with both need of daily insulin dose and the insulin-glucose product in non-diabetic septic cases (ptyrosine excretion, urinary meta-tyrosine/para-tyrosine, urinary ortho-tyrosine/para-tyrosine and urinary (meta- + orthotyrosine)/ para-tyrosine proved to be markers of carbohydrate homeostasis. In a chronic rodent model, we tried to compensate the abnormal tyrosine isomers using para-tyrosine, the physiological amino acid. Rats were fed a standard high cholesterol-diet, and were given para-tyrosine or vehicle orally. High-cholesterol feeding lead to a significant increase in aortic wall meta-tyrosine content and a decreased vasorelaxation of the aorta to insulin and the glucagon-like peptide-1 analogue, liraglutide, that both could be prevented by administration of para-tyrosine. Concluding, these data suggest that meta- and ortho-tyrosine are potential markers of oxidative stress in acute diseases related to oxidative stress, and may also interfere with insulin action in septic humans. Competition of meta- and ortho-tyrosine by supplementation of para-tyrosine may exert a protective role in oxidative stress-related diseases.

  2. High frequency of cytolytic 21-Hydroxylase specific CD8+ T cells in autoimmune Addison’s disease patients1

    Science.gov (United States)

    Dawoodji, Amina; Chen, Ji-Li; Shepherd, Dawn; Dalin, Frida; Tarlton, Andrea; Alimohammadi, Mohammad; Penna-Martinez, Marissa; Meyer, Gesine; Mitchell, Anna L; Gan, Earn H; Bratland, Eirik; Bensing, Sophie; Husebye, Eystein; Pearce, Simon H.; Badenhoop, Klaus; Kämpe, Olle; Cerundolo, Vincenzo

    2016-01-01

    The mechanisms behind the destruction of the adrenal glands in autoimmune Addison’s disease remain unclear. Autoantibodies against steroid 21-hydroxylase, an intracellular key enzyme of the adrenal cortex, are found in over 90% of patients, but these autoantibodies are not thought to mediate the disease. Here we demonstrate highly frequent 21-hydroxylase specific T cells detectable in 20 patients with Addison’s disease. Using overlapping 18aa peptides spanning the full length of 21-hydroxylase, we identified immunodominant CD8+ and CD4+ T cell responses in a large proportion of Addison’s patients both ex-vivo and after in-vitro culture of peripheral blood lymphocytes up to 20 years after diagnosis. In a large proportion of patients, CD8+ 21-hydroxylase specific T cells and CD4+ 21-hydroxylase specific T cells were very abundant and detectable in ex-vivo assays. HLA class-I tetramer-guided isolation of 21-hydroxylase specific CD8+ T cells showed their ability to lyse 21-hydroxylase positive target cells, consistent with a potential mechanism for disease pathogenesis. These data indicate strong cytotoxic T lymphocyte responses to 21-hydroxylase often occur in-vivo, and that reactive cytotoxic T lymphocytes have substantial proliferative and cytolytic potential. These results have implications for earlier diagnosis of adrenal failure and ultimately a potential target for therapeutic intervention and induction of immunity against adrenal cortex cancer. PMID:25063864

  3. Requirement for tyrosine phosphatase during serotonergic neuromodulation by protein kinase C.

    Science.gov (United States)

    Catarsi, S; Drapeau, P

    1997-08-01

    Tyrosine kinases and phosphatases are abundant in the nervous system, where they signal cellular differentiation, mediate the responses to growth factors, and direct neurite outgrowth during development. Tyrosine phosphorylation can also alter ion channel activity, but its physiological significance remains unclear. In an identified leech mechanosensory neuron, the ubiquitous neuromodulator serotonin increases the activity of a cation channel by activating protein kinase C (PKC), resulting in membrane depolarization and modulation of the receptive field properties. We observed that the effects on isolated neurons and channels were blocked by inhibiting tyrosine phosphatases. Serotonergic stimulation of PKC thus activates a tyrosine phosphatase activity associated with the channels, which reverses their constitutive inhibition by tyrosine phosphorylation, representing a novel form of neuromodulation.

  4. Overhydroxylation of Lysine of Collagen Increases Uterine Fibroids Proliferation: Roles of Lysyl Hydroxylases, Lysyl Oxidases, and Matrix Metalloproteinases

    Directory of Open Access Journals (Sweden)

    Marwa Kamel

    2017-01-01

    Full Text Available The role of the extracellular matrix (ECM in uterine fibroids (UF has recently been appreciated. Overhydroxylation of lysine residues and the subsequent formation of hydroxylysylpyridinoline (HP and lysylpyridinoline (LP cross-links underlie the ECM stiffness and profoundly affect tumor progression. The aim of the current study was to investigate the relationship between ECM of UF, collagen and collagen cross-linking enzymes [lysyl hydroxylases (LH and lysyl oxidases (LOX], and the development and progression of UF. Our results indicated that hydroxyl lysine (Hyl and HP cross-links are significantly higher in UF compared to the normal myometrial tissues accompanied by increased expression of LH (LH2b and LOX. Also, increased resistance to matrix metalloproteinases (MMP proteolytic degradation activity was observed. Furthermore, the extent of collagen cross-links was positively correlated with the expression of myofibroblast marker (α-SMA, growth-promoting markers (PCNA; pERK1/2; FAKpY397; Ki-67; and Cyclin D1, and the size of UF. In conclusion, our study defines the role of overhydroxylation of collagen and collagen cross-linking enzymes in modulating UF cell proliferation, differentiation, and resistance to MMP. These effects can establish microenvironment conducive for UF progression and thus represent potential target treatment options of UF.

  5. Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers

    Directory of Open Access Journals (Sweden)

    Pizzichini Daniele

    2007-03-01

    Full Text Available Abstract Background Beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein (in the beta-epsilon branch and violaxanthin (in the beta-beta branch. None of these carotenoids have provitamin A activity. We have previously shown that tuber-specific silencing of the first step in the epsilon-beta branch, LCY-e, redirects metabolic flux towards beta-beta carotenoids, increases total carotenoids up to 2.5-fold and beta-carotene up to 14-fold. Results In this work, we silenced the non-heme beta-carotene hydroxylases CHY1 and CHY2 in the tuber. Real Time RT-PCR measurements confirmed the tuber-specific silencing of both genes . CHY silenced tubers showed more dramatic changes in carotenoid content than LCY-e silenced tubers, with beta-carotene increasing up to 38-fold and total carotenoids up to 4.5-fold. These changes were accompanied by a decrease in the immediate product of beta-carotene hydroxylation, zeaxanthin, but not of the downstream xanthophylls, viola- and neoxanthin. Changes in endogenous gene expression were extensive and partially overlapping with those of LCY-e silenced tubers: CrtISO, LCY-b and ZEP were induced in both cases, indicating that they may respond to the balance between individual carotenoid species. Conclusion Together with epsilon-cyclization of lycopene, beta-carotene hydroxylation is another regulatory step in potato tuber carotenogenesis. The data are consistent with a prevalent role of CHY2, which is highly expressed in tubers, in the control of this step. Combination of different engineering strategies holds good promise for the manipulation of tuber carotenoid content.

  6. Conformational Clusters of Phosphorylated Tyrosine.

    Science.gov (United States)

    Abdelrasoul, Maha; Ponniah, Komala; Mao, Alice; Warden, Meghan S; Elhefnawy, Wessam; Li, Yaohang; Pascal, Steven M

    2017-12-06

    Tyrosine phosphorylation plays an important role in many cellular and intercellular processes including signal transduction, subcellular localization, and regulation of enzymatic activity. In 1999, Blom et al., using the limited number of protein data bank (PDB) structures available at that time, reported that the side chain structures of phosphorylated tyrosine (pY) are partitioned into two conserved conformational clusters ( Blom, N.; Gammeltoft, S.; Brunak, S. J. Mol. Biol. 1999 , 294 , 1351 - 1362 ). We have used the spectral clustering algorithm to cluster the increasingly growing number of protein structures with pY sites, and have found that the pY residues cluster into three distinct side chain conformations. Two of these pY conformational clusters associate strongly with a narrow range of tyrosine backbone conformation. The novel cluster also highly correlates with the identity of the n + 1 residue, and is strongly associated with a sequential pYpY conformation which places two adjacent pY side chains in a specific relative orientation. Further analysis shows that the three pY clusters are associated with distinct distributions of cognate protein kinases.

  7. 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

  8. Dose-dependent effects of oral tyrosine administration on plasma tyrosine levels and cognition in aging

    NARCIS (Netherlands)

    Rest, van de Ondine; Bloemendaal, Mirjam; Heus, De Rianne; Aarts, Esther

    2017-01-01

    The effects of tyrosine on plasma response and cognition in aging are unknown. We assessed the dose-dependent response to tyrosine administration in older adults in both plasma tyrosine concentrations and working memory performance. In this double blind randomized cross-over trial 17 older adults

  9. Dose-Dependent Effects of Oral Tyrosine Administration on Plasma Tyrosine Levels and Cognition in Aging

    NARCIS (Netherlands)

    Rest, O. van de; Bloemendaal, M.; Heus, R.A.A. de; Aarts, E.

    2017-01-01

    The effects of tyrosine on plasma response and cognition in aging are unknown. We assessed the dose-dependent response to tyrosine administration in older adults in both plasma tyrosine concentrations and working memory performance. In this double blind randomized cross-over trial 17 older adults

  10. Catecholaminergic development of fetal rat ventral mesencephalon : Characterization by high-performance liquid chromatography with electrochemical detection and immunohistochemistry

    NARCIS (Netherlands)

    Tomasini, R; Kema, IP; Muskiet, FAJ; Meiborg, G; Staal, MJ; Go, KG

    We determined dopamine (DA), noradrenaline (NA), and adrenaline (A), as well as immunohistochemically stained tyrosine hydroxylase (TH) and DA in dissected rat ventral mesencephalon (VM) tissue from Embryonic Day (ED) 14 to Postnatal Day (P) 17. Whole VM tissue DA, NA, and A contents increased with

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

    Science.gov (United States)

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

    2017-03-07

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

  12. 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.

  13. 21 CFR 582.5920 - Tyrosine.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Tyrosine. 582.5920 Section 582.5920 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS... § 582.5920 Tyrosine. (a) Product. Tyrosine (L- and DL-forms). (b) Conditions of use. This substance is...

  14. Tyrosine dephosphorylation regulates AMPAR internalisation in mGluR-LTD.

    Science.gov (United States)

    Gladding, Clare M; Collett, Valerie J; Jia, Zhengping; Bashir, Zafar I; Collingridge, Graham L; Molnár, Elek

    2009-02-01

    Long-term depression (LTD) can be induced at hippocampal CA1 synapses by activation of either NMDA receptors (NMDARs) or group I metabotropic glutamate receptors (mGluRs), using their selective agonists NMDA and (RS)-3,5-dihydroxyphenylglycine (DHPG), respectively. Recent studies revealed that DHPG-LTD is dependent on activation of postsynaptic protein tyrosine phosphatases (PTPs), which transiently dephosphorylate tyrosine residues in AMPA receptors (AMPARs). Here we show that while both endogenous GluR2 and GluR3 AMPAR subunits are tyrosine phosphorylated at basal activity, only GluR2 is dephosphorylated in DHPG-LTD. The tyrosine dephosphorylation of GluR2 does not occur in NMDA-LTD. Conversely, while NMDA-LTD is associated with the dephosphorylation of GluR1-serine-845, DHPG-LTD does not alter the phosphorylation of this site. The increased AMPAR endocytosis in DHPG-LTD is PTP-dependent and involves tyrosine dephosphorylation of cell surface AMPARs. Together, these results indicate that the subunit selective tyrosine dephosphorylation of surface GluR2 regulates AMPAR internalisation in DHPG-LTD but not in NMDA-LTD in the hippocampus.

  15. Assays for mammalian tyrosinase: a comparative study

    International Nuclear Information System (INIS)

    Jara, J.R.; Solano, F.; Lozano, J.A.

    1988-01-01

    This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed

  16. 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.

  17. Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 2. Tyrosine-26 and -64

    International Nuclear Information System (INIS)

    Roepe, P.; Scherrer, P.; Ahl, P.L.; Gupta, S.K.D.; Bogomolni, R.A.; Herzfeld, J.; Rothschild, K.J.

    1987-01-01

    Low-temperature Fourier transform infrared (FTIR) and UV difference spectroscopies combined with selective tyrosine nitration and tyrosine isotopic labeling have been used to investigate the participation of tyrosines-26 and -64 in the bacteriorhodopsin (bR) photocycle. Nitration of Tyr-26 has no detectable effect on the FTIR or UV difference spectra of the BR 570 → K 630 or BR 570 → M 412 transitions. In contrast, nitration of Tyr-64 causes changes in both the FTIR and UV spectra of these transitions. However, this nitration does not alter tyrosine peaks in the FTIR difference spectra which have previously been associated with the protonation of a tyrosinate by K 630 and the deprotonation of a tyrosine by M 412 . Instead, Tyr-64 nitration appears to affect other tyrosine peaks. These results and changes in UV difference spectra upon Tyr-64 nitration are consistent with the deprotonation of Tyr-64 by M 412 as concluded previously. Effects on chromophore vibrations caused by Tyr-64 nitration are unaltered upon reducing the nitrotyrosine to aminotyrosine with sodium dithionite. Finally, nitro-Tyr-64 causes a shift in the frequency of a positive peak at 1739 cm -1 in the BR 570 → M 412 FTIR difference spectrum which reflects the protonation of a carboxyl-containing residue. The shift does not occur for samples containing amino-Tyr-64. These data suggest that Tyr-64 may interact with this carboxyl group

  18. Mechanism of papain-catalyzed synthesis of oligo-tyrosine peptides.

    Science.gov (United States)

    Mitsuhashi, Jun; Nakayama, Tsutomu; Narai-Kanayama, Asako

    2015-01-01

    Di-, tri-, and tetra-tyrosine peptides with angiotensin I-converting enzyme inhibitory activity were synthesized by papain-catalyzed polymerization of L-tyrosine ethyl ester in aqueous media at 30 °C. Varying the reaction pH from 6.0 to 7.5 and the initial concentration of the ester substrate from 25 to 100 mM, the highest yield of oligo-tyrosine peptides (79% on a substrate basis) was produced at pH 6.5 and 75 mM, respectively. In the reaction initiated with 100 mM of the substrate, approx. 50% yield of insoluble, highly polymerized peptides accumulated. At less than 15 mM, the reaction proceeded poorly; however, from 30 mM to 120 mM a dose-dependent increase in the consumption rate of the substrate was observed with a sigmoidal curve. Meanwhile, each of the tri- and tetra-tyrosine peptides, even at approx. 5mM, was consumed effectively by papain but was not elongated to insoluble polymers. For deacylation of the acyl-papain intermediate through which a new peptide bond is made, L-tyrosine ethyl ester, even at 5mM, showed higher nucleophilic activity than di- and tri-tyrosine. These results indicate that the mechanism through which papain polymerizes L-tyrosine ethyl ester is as follows: the first interaction between papain and the ester substrate is a rate-limiting step; oligo-tyrosine peptides produced early in the reaction period are preferentially used as acyl donors, while the initial ester substrate strongly contributes as a nucleophile to the elongation of the peptide product; and the balance between hydrolytic fragmentation and further elongation of oligo-tyrosine peptides is dependent on the surrounding concentration of the ester substrate. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity.

    Science.gov (United States)

    Argetsinger, Lawrence S; Kouadio, Jean-Louis K; Steen, Hanno; Stensballe, Allan; Jensen, Ole N; Carter-Su, Christin

    2004-06-01

    The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 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 decreasing kinase activity and thereby contributing to rapid termination of ligand activation of JAK2.

  20. Midbrain expression of Delta-like 1 homologue is regulated by GDNF and is associated with dopaminergic differentiation

    DEFF Research Database (Denmark)

    Christophersen, Nicolai S.; Gronborg, Mette; Petersen, Thomas Nordahl

    2007-01-01

    upregulation with increased positive staining of cell bodies in the SNc and fibers in the striatum. Analysis of the developmental regulation of Dlk1 in the murine ventral midbrain showed that the upregulation of Dlk1 mRNA correlated with the generation of tyrosine hydroxylase (TH)-positive neurons. Furthermore...

  1. Cerebral 5-HT2A receptor binding, but not mGluR2, is increased in tryptophan hydroxylase 2 decrease-of-function mice

    DEFF Research Database (Denmark)

    Jørgensen, Christinna Vangsgaard; Jacobsen, Jacob P; Caron, Marc G

    2013-01-01

    Transgenic mice with a knock-in (KI) of a tryptophan hydroxylase 2 (Tph2) R439H mutation, analogous to the Tph2 R441H single-nucleotide polymorphism originally identified in a late life depression cohort, have markedly reduced levels of 5-hydroxytryptamine (5-HT). These Tph2KI mice are therefore...

  2. Structural Insights into Diversity and n-Alkane Biodegradation Mechanisms of Alkane Hydroxylases

    Directory of Open Access Journals (Sweden)

    Yurui eJi

    2013-03-01

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

  3. 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

  4. Origin and characterization of retrograde labeled neurons supplying the rat urethra using fiberoptic confocal fluorescent microscopy in vivo and immunohistochemistry.

    Science.gov (United States)

    Lee, Keon-Cheol; Sharma, Seema; Tuttle, Jeremy B; Steers, William D

    2010-10-01

    Autonomic innervation of urethral smooth muscle may influence urinary continence after prostatectomy. It is unclear whether the cavernous nerves carry fibers that influence continence. Using a retrograde axonal tracer combined with real-time in vivo imaging and ex vivo immunohistochemistry we determined the course and type of neurons supplying urethral smooth muscle distal to the prostate in the rat. We injected the retrograde axonal tracers cholera toxin B fragment-Alexa Fluor 488 and Fast Blue in the distal urethral smooth muscle in 10 rats each. Five days later the cavernous nerves and pelvic ganglion were imaged using fiberoptic confocal fluorescence microscopy (cholera toxin B fragment-Alexa Fluor 488) or harvested for immunohistochemistry (Fast Blue). Dual immunofluorescence of Fast Blue neurons with tyrosine hydroxylase or neuronal nitric oxide synthase was done to characterize neurons as noradrenergic or nitrergic. To ascertain whether the cavernous nerves contain fibers to the urethra that originate in the pelvic ganglia we cut the cavernous nerves with their ancillary branches in 3 rats and imaged them for Fast Blue. Fluorescent neurons and axons were detected in cavernous nerves and the pelvic ganglion. Few neurons were seen in rats with cavernous nerve section. Of urethral neurons 53.1% showed neuronal nitric oxide synthase positivity while 40.6% were immunoreactive for tyrosine hydroxylase. About 6.2% of urethral neurons failed to show tyrosine hydroxylase or neuronal nitric oxide synthase immunoreactivity. Most of the autonomic innervation to the urethra beyond the prostatic apex travels in the cavernous nerves. Many nerves may be parasympathetic based on neuronal nitric oxide synthase immunoreactivity. Nerves supplying the urethra outside the cavernous nerves may course posterior to the prostate. Along with afferent fibers, tyrosine hydroxylase immunoreactivity expressing neuron fibers, ie noradrenergic nerves, traveling in the cavernous nerves may

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

    Science.gov (United States)

    Shak, S; Goldstein, I M

    1985-09-01

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

  6. 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...

  7. A potential benefit of albinism in Astyanax cavefish: downregulation of the oca2 gene increases tyrosine and catecholamine levels as an alternative to melanin synthesis.

    Science.gov (United States)

    Bilandžija, Helena; Ma, Li; Parkhurst, Amy; Jeffery, William R

    2013-01-01

    Albinism, the loss of melanin pigmentation, has evolved in a diverse variety of cave animals but the responsible evolutionary mechanisms are unknown. In Astyanax mexicanus, which has a pigmented surface dwelling form (surface fish) and several albino cave-dwelling forms (cavefish), albinism is caused by loss of function mutations in the oca2 gene, which operates during the first step of the melanin synthesis pathway. In addition to albinism, cavefish have evolved differences in behavior, including feeding and sleep, which are under the control of the catecholamine system. The catecholamine and melanin synthesis pathways diverge after beginning with the same substrate, L-tyrosine. Here we describe a novel relationship between the catecholamine and melanin synthesis pathways in Astyanax. Our results show significant increases in L-tyrosine, dopamine, and norepinephrine in pre-feeding larvae and adult brains of Pachón cavefish relative to surface fish. In addition, norepinephrine is elevated in cavefish adult kidneys, which contain the teleost homologs of catecholamine synthesizing adrenal cells. We further show that the oca2 gene is expressed during surface fish development but is downregulated in cavefish embryos. A key finding is that knockdown of oca2 expression in surface fish embryos delays the development of pigmented melanophores and simultaneously increases L-tyrosine and dopamine. We conclude that a potential evolutionary benefit of albinism in Astyanax cavefish may be to provide surplus L-tyrosine as a precursor for the elevated catecholamine synthesis pathway, which could be important for adaptation to the challenging cave environment.

  8. A potential benefit of albinism in Astyanax cavefish: downregulation of the oca2 gene increases tyrosine and catecholamine levels as an alternative to melanin synthesis.

    Directory of Open Access Journals (Sweden)

    Helena Bilandžija

    Full Text Available Albinism, the loss of melanin pigmentation, has evolved in a diverse variety of cave animals but the responsible evolutionary mechanisms are unknown. In Astyanax mexicanus, which has a pigmented surface dwelling form (surface fish and several albino cave-dwelling forms (cavefish, albinism is caused by loss of function mutations in the oca2 gene, which operates during the first step of the melanin synthesis pathway. In addition to albinism, cavefish have evolved differences in behavior, including feeding and sleep, which are under the control of the catecholamine system. The catecholamine and melanin synthesis pathways diverge after beginning with the same substrate, L-tyrosine. Here we describe a novel relationship between the catecholamine and melanin synthesis pathways in Astyanax. Our results show significant increases in L-tyrosine, dopamine, and norepinephrine in pre-feeding larvae and adult brains of Pachón cavefish relative to surface fish. In addition, norepinephrine is elevated in cavefish adult kidneys, which contain the teleost homologs of catecholamine synthesizing adrenal cells. We further show that the oca2 gene is expressed during surface fish development but is downregulated in cavefish embryos. A key finding is that knockdown of oca2 expression in surface fish embryos delays the development of pigmented melanophores and simultaneously increases L-tyrosine and dopamine. We conclude that a potential evolutionary benefit of albinism in Astyanax cavefish may be to provide surplus L-tyrosine as a precursor for the elevated catecholamine synthesis pathway, which could be important for adaptation to the challenging cave environment.

  9. 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

  10. Allosteric activation of midazolam CYP3A5 hydroxylase activity by icotinib - Enhancement by ketoconazole.

    Science.gov (United States)

    Zhuang, XiaoMei; Zhang, TianHong; Yue, SiJia; Wang, Juan; Luo, Huan; Zhang, YunXia; Li, Zheng; Che, JinJing; Yang, HaiYing; Li, Hua; Zhu, MingShe; Lu, Chuang

    2016-12-01

    Icotinib (ICO), a novel small molecule and a tyrosine kinase inhibitor, was developed and approved recently in China for non-small cell lung cancer. During screening for CYP inhibition potential in human liver microsomes (HLM), heterotropic activation toward CYP3A5 was revealed. Activation by icotinib was observed with CYP3A-mediated midazolam hydroxylase activity in HLM (∼40% over the baseline) or recombinant human CYP3A5 (rhCYP3A5) (∼70% over the baseline), but not in the other major CYPs including rhCYP3A4. When co-incubated with selective CYP3A4 inhibitor CYP3cide or monoclonal human CYP3A4 inhibitory antibody in HLM, the activation was extended to ∼60%, suggesting CYP3A5 might be the isozyme involved. Further, the relative activation was enhanced to ∼270% in rhCYP3A5 in the presence of ketoconazole. The activation was substrate and pathway dependent and observed only in the formation of 1'-OH-midazolam, and not 4-OH-midazolam, 6β-OH-testosterone, or oxidized nifedipine. The activation requires the presence of cytochrome b5 and it is only observed in the liver microsomes of dogs, monkeys, and humans, but not in rats and mice. Kinetic analyses of 1'-OH-midazolam formation showed that ICO increased the V max values in HLM and rhCYP3A5 with no significant changes in K m values. By adding CYP3cide with ICO to the incubation, the V max values increased 2-fold over the CYP3cide control. Addition of ketoconazole with ICO alone or ICO plus CYP3cide resulted in an increase in V max values and decrease in K m values compared to their controls. This phenomenon may be attributed to a new mechanism of CYP3A5 heterotropic activation, which warrants further investigation. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Fermented soymilk increases voluntary wheel running activity and sexual behavior in male rats.

    Science.gov (United States)

    Sato, Takuya; Shinohara, Yasutomo; Kaneko, Daisuke; Nishimura, Ikuko; Matsuyama, Asahi

    2010-12-01

    Wheel running by rodents is thought to reflect voluntary exercise in humans. The present study examined the effect of fermented soymilk (FSM) on voluntary wheel running in rats. FSM was prepared from soymilk (SM) using the bacteria Leuconostoc pseudomesenteroides. The rats were fed a normal diet for 3 weeks followed by a 3-week administration of diet containing FSM or SM (5% w/w), and then the diets were switched back to a normal diet for 3 weeks. The voluntary wheel running activity was increased by FSM administration, although no changes were observed by SM administration. This effect was observed 2 weeks after FSM administration and lasted 1 week after deprivation of FSM. Then we evaluated the effect of FSM on sexual behavior in male rats. FSM administration for 10 days significantly increased the number of mounts. The protein expression of tyrosine hydroxylase (TH) increased in the hippocampus by FSM administration and it is suggested that FSM may change norepinephrine or dopamine signaling in the brain. Our study provides the first evidence that FSM increases voluntary wheel running activity and sexual behavior and suggests that TH may be involved in these effects.

  12. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Mostafa, M.H.; Sheweita, S.A.; Abdel-Moneam, N.M. (Alexandria Univ. (Egypt))

    1990-06-01

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

  14. 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....

  15. Effects of chemical sympathectomy on the increases in plasma catecholamines and dopamine-beta-hydroxylase induced by forced immobilization and insulin-induced hypoglycemia: origin and fate of plasma dopamine-beta-hydroxylase.

    Science.gov (United States)

    Israel, A S; Barbella, Y R; Cubeddu, L X

    1982-06-01

    The effect of acute stresses on plasma norepinephrine, epinephrine and dopamine-beta-hydroxylase (DBH) were evaluated in control and 6-hydroxydopamine-treated, awake cannulated guinea pigs. Forced immobolization for 1 hr caused a 3- and 5-fold increase in plasma DBH and norepinephrine, respectively. Pretreatment with 6-hydroxydopamine (23 mg/kg b.wt.i.a., 72 and 48 hr before stress) reduced by 70% the increase in plasma DBH and totally prevented the rise in plasma catecholamines evoked by the restraining stress. Injection of insulin (5 U/kg b.wt.i.a.) induced a 60% decrease in blood glucose, a 1-fold increase in plasma DBH and a selective 4-fold increase in plasma epinephrine; these effects were not modified by chemical sympathectomy. Our results indicate that forced immobilization and hypoglycemia produce a preferential activation of the sympathetic postganglionic nerves and of the adrenal medulla, respectively, and that in guinea pigs both stresses increase plasma DBH. The kinetics of disappearance of plasma DBH were studied after subjecting the guinea pigs for 1 hr to forced immobilization. Although 7 of 12 animals showed a biphasic rate of fall of plasma DBH, in each case there was a rapid initial fall possibly due to the "distribution" of the enzyme with a T1/2 of 1.65 hr. Similar findings were observed in 6-hydroxydopamine-treated guinea pigs. These results suggest that the distribution of DBH is the most important process in reducing the augmented plasma DBH levels elicited by a short-term stress and that this process is not dependent on the integrity of the sympathetic nerves nor on the adrenal or sympathetic origin of the enzyme. This study supports the view that the ratio, content of releasable DBH present in sympathetic nerves and adrenal glands/total circulating pool of DBH, is the factor that determines whether an increase in plasma DBH would occur in animals exposed to an acute stress.

  16. 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

  17. Role of tyrosine phosphatase inhibitors in cancer treatment with emphasis on SH2 domain-containing tyrosine phosphatases (SHPs)

    NARCIS (Netherlands)

    Irandoust, Mahban; van den Berg, Timo K.; Kaspers, Gertjan J. L.; Cloos, Jacqueline

    2009-01-01

    Protein tyrosine phosphorylation is one of the key mechanisms involved in signal transduction pathways. This modification is regulated by concerted action of protein tyrosine phosphatases and protein tyrosine kinases. Deregulation of either of these key regulators lead to abnormal cellular

  18. Nogo-receptor 1 antagonization in combination with neurotrophin-4/5 is not superior to single factor treatment in promoting survival and morphological complexity of cultured dopaminergic neurons.

    Science.gov (United States)

    Seiler, Stefanie; Di Santo, Stefano; Sahli, Sebastian; Andereggen, Lukas; Widmer, Hans Rudolf

    2017-08-01

    Cell transplantation using ventral mesencephalic tissue is an experimental approach to treat Parkinson's disease. This approach is limited by poor survival of the transplants and the high number of dopaminergic neurons needed for grafting. Increasing the yield of dopaminergic neurons in donor tissue is of great importance. We have previously shown that antagonization of the Nogo-receptor 1 by NEP1-40 promoted survival of cultured dopaminergic neurons and exposure to neurotrophin-4/5 increased dopaminergic cell densities in organotypic midbrain cultures. We investigated whether a combination of both treatments offers a novel tool to further improve dopaminergic neuron survival. Rat embryonic ventral mesencephalic neurons grown as organotypic free-floating roller tube or primary dissociated cultures were exposed to neurotrophin-4/5 and NEP1-40. The combined and single factor treatment resulted in significantly higher numbers of tyrosine hydroxylase positive neurons compared to controls. Significantly stronger tyrosine hydroxylase signal intensity was detected by Western blotting in the combination-treated cultures compared to controls but not compared to single factor treatments. Neurotrophin-4/5 and the combined treatment showed significantly higher signals for the neuronal marker microtubule-associated protein 2 in Western blots compared to control while no effects were observed for the astroglial marker glial fibrillary acidic protein between groups, suggesting that neurotrophin-4/5 targets mainly neuronal cells. Finally, NEP1-40 and the combined treatment significantly augmented tyrosine hydroxylase positive neurite length. Summarizing, our findings substantiate that antagonization of the Nogo-receptor 1 promotes dopaminergic neurons but does not further increase the yield of dopaminergic neurons and their morphological complexity when combined with neurotrophin-4/5 hinting to the idea that these treatments might exert their effects by activating common

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

    Science.gov (United States)

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

    2005-10-01

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

  20. Genesis by meiotic unequal crossover of a de novo deletion that contributes to steroid 21-hydroxylase deficiency

    International Nuclear Information System (INIS)

    Sinnott, P.; Collier, S.; Dyer, P.A.; Harris, R.; Strachan, T.; Costigan, C.

    1990-01-01

    The HLA-linked human steroid 21-hydroxylase gene CYP21B and its closely homologous pseudogene CYP21A are each normally located centromeric to a fourth component of complement (C4) gene, C4B and C4A, respectively, in an organization suggesting tandem duplication of a ca. 30-kilobase DNA unit containing a CYP21 gene and a C4 gene. Such an organization has been considered to facilitate gene deletion and addition events by unequal crossover between the tandem repeats. The authors have identified a steroid 21-hydroxylase deficiency patient who has a maternally inherited disease haplotype that carries a de novo deletion of a ca. 30-kilobase repeat unit including the CYP21B gene and associated C4B gene. This disease haplotype appears to have been generated as a result of meiotic unequal crossover between maternal homologous chromosomes. One of the maternal haplotypes is the frequently occurring HLA-DR3,B8,A1 haplotype that normally carries a deletion of a ca. 30-kilobase unit including the CYP21A gene and C4A gene. Haplotypes of this type may possible act as premutations, increasing the susceptibility of developing a 21-hydroxylase deficiency mutation by facilitating unequal chromosome pairing

  1. 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.

  2. Synthesis of deuterium and tritium labelled tyrosine

    International Nuclear Information System (INIS)

    Kanska, M.; Drabarek, S.

    1980-01-01

    A new method of synthesis of tyrosine labelled with deuterium and tritium in the aromatic ring has been developed. Deuterated and tritiated tyrosine was obtained by isotope exchange between tyrosine and deuterated or tritiated water at elevated temperature in hydrochloric acid medium using K 2 PtCl 4 as a catalyst. For synthesis of tritiated tyrosine 1 Ci HTO was used; the specific activity of the product was 5 mCi/mMol. (author)

  3. Tyrosine-sensitized photodimerization of thymine in aqueous solution

    International Nuclear Information System (INIS)

    Kaneko, M.; Matsuyama, A.; Nagata, C.

    1978-01-01

    Photodimerization of thymine in aqueous solution in the presence of tyrosine was studied with monochromatic UV irradiation. The total dimer formation was sensitized in the presence of tyrosine. The action spectrum of sensitized total dimer formation has a peak near 280 nm corresponding to the absorption maximum of tyrosine. Triplet quenchers reduced the sensitization substantially. It seems probable that tyrosine-sensitized photodimerization of thymine occurred via triplet-triplet energy transfer from tyrosine to thymine. (author)

  4. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

  5. e-Cadherin in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-Induced Parkinson Disease

    Directory of Open Access Journals (Sweden)

    Samuela Cataldi

    2016-01-01

    Full Text Available Today a large number of studies are focused on clarifying the complexity and diversity of the pathogenetic mechanisms inducing Parkinson disease. We used 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, a neurotoxin that induces Parkinson disease, to evaluate the change of midbrain structure and the behavior of the anti-inflammatory factor e-cadherin, interleukin-6, tyrosine hydroxylase, phosphatase and tensin homolog, and caveolin-1. The results showed a strong expression of e-cadherin, variation of length and thickness of the heavy neurofilaments, increase of interleukin-6, and reduction of tyrosine hydroxylase known to be expression of dopamine cell loss, reduction of phosphatase and tensin homolog described to impair responses to dopamine, and reduction of caveolin-1 known to be expression of epithelial-mesenchymal transition and fibrosis. The possibility that the overexpression of the e-cadherin might be implicated in the anti-inflammatory reaction to MPTP treatment by influencing the behavior of the other analyzed molecules is discussed.

  6. 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.

  7. 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.

  8. Effect of tyrosine administration on duodenal ulcer induced by cysteamine in the rat

    International Nuclear Information System (INIS)

    Oishi, T.; Szabo, S.

    1987-01-01

    Duodenal ulcers were produced by administering cysteamine to rats. Pretreatment with the catecholamine precursor, L-tyrosine (40 mg/100 g i.p. for 5 days), decreased the intensity of duodenal ulcers induced by cysteamine. Equimolar doses of tyrosine methyl ester (51.2 mg/100 g i.p. or s.c.) were equally effective in reducing ulcer intensity. Other amino acids (i.e., alanine, aspartic acid, glutamic acid, glycine, leucine, lysine, tryptophan and valine) did not prevent experimental duodenal ulcers. Coadministration of other large neutral amino acids (e.g., leucine and valine) that compete with tyrosine for uptake into the brain did not inhibit the effect of tyrosine on duodenal ulcers induced by cysteamine. Gastric, duodenal and brain dopamine concentrations were increased 1 hr after the injection of tyrosine methyl ester (25.6 mg/100 g s.c.). These results suggest that the effect of tyrosine on duodenal ulcer induced by cysteamine may be mediated by changes in gastrointestinal dopamine metabolism

  9. 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

  10. 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...

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

    Science.gov (United States)

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

    1995-01-01

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

  12. 21 CFR 862.1730 - Free tyrosine test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Free tyrosine test system. 862.1730 Section 862....1730 Free tyrosine test system. (a) Identification. A free tyrosine test system is a device intended to measure free tyrosine (an amono acid) in serum and urine. Measurements obtained by this device are used in...

  13. Suppression of sterol 27-hydroxylase mRNA and transcriptional activity by bile acids in cultured rat hepatocytes

    NARCIS (Netherlands)

    Twisk, J.; Wit, E.C.M. de; Princen, H.M.G.

    1995-01-01

    In previous work we have demonstrated suppression of cholesterol 7α-hydroxylase by bile acids at the level of mRNA and transcription, resulting in a similar decline in bile acid synthesis in cultured rat hepatocytes. In view of the substantial contribution of the 'alternative' or '27-hydroxylase'

  14. 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

  15. 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.

  16. Characterization of carotenoid hydroxylase gene promoter in Haematococcus pluvialis.

    Science.gov (United States)

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

    2006-10-01

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

  17. Co-downregulation of the hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase and coumarate 3-hydroxylase significantly increases cellulose content in transgenic alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Tong, Zongyong; Li, Heng; Zhang, Rongxue; Ma, Lei; Dong, Jiangli; Wang, Tao

    2015-10-01

    Lignin is a component of the cell wall that is essential for growth, development, structure and pathogen resistance in plants, but high lignin is an obstacle to the conversion of cellulose to ethanol for biofuel. Genetically modifying lignin and cellulose contents can be a good approach to overcoming that obstacle. Alfalfa (Medicago sativa L.) is rich in lignocellulose biomass and used as a model plant for the genetic modification of lignin in this study. Two key enzymes in the lignin biosynthesis pathway-hydroxycinnamoyl -CoA:shikimate hydroxycinnamoyl transferase (HCT) and coumarate 3-hydroxylase (C3H)-were co-downregulated. Compared to wild-type plants, the lignin content in the modified strain was reduced by 38%, cellulose was increased by 86.1%, enzyme saccharification efficiency was increased by 10.9%, and cell wall digestibility was increased by 13.0%. The modified alfalfa exhibited a dwarf phenotype, but normal above ground biomass. This approach provides a new strategy for reducing lignin and increasing cellulose contents and creates a new genetically modified crop with enhanced value for biofuel. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

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

    1979-07-01

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

  19. Tyrosine kinase inhibition increases the cell surface localization of FLT3-ITD and enhances FLT3-directed immunotherapy of acute myeloid leukemia.

    Science.gov (United States)

    Reiter, K; Polzer, H; Krupka, C; Maiser, A; Vick, B; Rothenberg-Thurley, M; Metzeler, K H; Dörfel, D; Salih, H R; Jung, G; Nößner, E; Jeremias, I; Hiddemann, W; Leonhardt, H; Spiekermann, K; Subklewe, M; Greif, P A

    2018-02-01

    The fms-related tyrosine kinase 3 (FLT3) receptor has been extensively studied over the past two decades with regard to oncogenic alterations that do not only serve as prognostic markers but also as therapeutic targets in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) became of special interest in this setting as they are associated with unfavorable prognosis. Because of sequence-dependent protein conformational changes FLT3-ITD tends to autophosphorylate and displays a constitutive intracellular localization. Here, we analyzed the effect of tyrosine kinase inhibitors (TKIs) on the localization of the FLT3 receptor and its mutants. TKI treatment increased the surface expression through upregulation of FLT3 and glycosylation of FLT3-ITD and FLT3-D835Y mutants. In T cell-mediated cytotoxicity (TCMC) assays, using a bispecific FLT3 × CD3 antibody construct, the combination with TKI treatment increased TCMC in the FLT3-ITD-positive AML cell lines MOLM-13 and MV4-11, patient-derived xenograft cells and primary patient samples. Our findings provide the basis for rational combination of TKI and FLT3-directed immunotherapy with potential benefit for FLT3-ITD-positive AML patients.

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

    Science.gov (United States)

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

    1977-01-01

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

  1. 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.

  2. Elevated levels of plasma phenylalanine in schizophrenia: a guanosine triphosphate cyclohydrolase-1 metabolic pathway abnormality?

    Directory of Open Access Journals (Sweden)

    Olaoluwa Okusaga

    Full Text Available BACKGROUND: Phenylalanine and tyrosine are precursor amino acids required for the synthesis of dopamine, the main neurotransmitter implicated in the neurobiology of schizophrenia. Inflammation, increasingly implicated in schizophrenia, can impair the function of the enzyme Phenylalanine hydroxylase (PAH; which catalyzes the conversion of phenylalanine to tyrosine and thus lead to elevated phenylalanine levels and reduced tyrosine levels. This study aimed to compare phenylalanine, tyrosine, and their ratio (a proxy for PAH function in a relatively large sample of schizophrenia patients and healthy controls. METHODS: We measured non-fasting plasma phenylalanine and tyrosine in 950 schizophrenia patients and 1000 healthy controls. We carried out multivariate analyses to compare log transformed phenylalanine, tyrosine, and phenylalanine:tyrosine ratio between patients and controls. RESULTS: Compared to controls, schizophrenia patients had higher phenylalanine (p<0.0001 and phenylalanine: tyrosine ratio (p<0.0001 but tyrosine did not differ between the two groups (p = 0.596. CONCLUSIONS: Elevated phenylalanine and phenylalanine:tyrosine ratio in the blood of schizophrenia patients have to be replicated in longitudinal studies. The results may relate to an abnormal PAH function in schizophrenia that could become a target for novel preventative and interventional approaches.

  3. Skin peptide tyrosine-tyrosine, a member of the pancreatic polypeptide family: isolation, structure, synthesis, and endocrine activity.

    Science.gov (United States)

    Mor, A; Chartrel, N; Vaudry, H; Nicolas, P

    1994-10-25

    Pancreatic polypeptide, peptide tyrosine-tyrosine (PYY), and neuropeptide tyrosine (NPY), three members of a family of structurally related peptides, are mainly expressed in the endocrine pancreas, in endocrine cells of the gut, and in the brain, respectively. In the present study, we have isolated a peptide of the pancreatic polypeptide family from the skin of the South American arboreal frog Phyllomedusa bicolor. The primary structure of the peptide was established as Tyr-Pro-Pro-Lys-Pro-Glu-Ser-Pro-Gly-Glu10-Asp-Ala-Ser-Pro-Glu-Glu- Met-Asn- Lys-Tyr20-Leu-Thr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Val-Thr- Arg-Gln-Arg-Tyr-NH2 . This unusual peptide, named skin peptide tyrosine-tyrosine (SPYY), exhibits 94% similarity with PYY from the frog Rana ridibunda. A synthetic replicate of SPYY inhibits melanotropin release from perifused frog neurointermediate lobes in very much the same way as NPY. These results demonstrate the occurrence of a PYY-like peptide in frog skin. Our data also suggest the existence of a pituitary-skin regulatory loop in amphibians.

  4. Oxidative stress and regulation of Pink1 in zebrafish (Danio rerio.

    Directory of Open Access Journals (Sweden)

    Madhusmita Priyadarshini

    Full Text Available Oxidative stress-mediated neuronal dysfunction is characteristic of several neurodegenerative disorders, including Parkinson's disease (PD. The enzyme tyrosine hydroxylase (TH catalyzes the formation of L-DOPA, the rate-limiting step in the biosynthesis of dopamine. A lack of dopamine in the striatum is the most characteristic feature of PD, and the cause of the most dominant symptoms. Loss of function mutations in the PTEN-induced putative kinase (PINK1 gene cause autosomal recessive PD. This study explored the basic mechanisms underlying the involvement of pink1 in oxidative stress-mediated PD pathology using zebrafish as a tool. We generated a transgenic line, Tg(pink1:EGFP, and used it to study the effect of oxidative stress (exposure to H2O2 on pink1 expression. GFP expression was enhanced throughout the brain of zebrafish larvae subjected to oxidative stress. In addition to a widespread increase in pink1 mRNA expression, mild oxidative stress induced a clear decline in tyrosine hydroxylase 2 (th2, but not tyrosine hydroxylase 1 (th1 expression, in the brain of wild-type larvae. The drug L-Glutathione Reduced (LGR has been associated with anti-oxidative and possible neuroprotective properties. Administration of LGR normalized the increased fluorescence intensity indicating pink1 transgene expression and endogenous pink1 mRNA expression in larvae subjected to oxidative stress by H2O2. In the pink1 morpholino oliogonucleotide-injected larvae, the reduction in the expression of th1 and th2 was partially rescued by LGR. The pink1 gene is a sensitive marker of oxidative stress in zebrafish, and LGR effectively normalizes the consequences of mild oxidative stress, suggesting that the neuroprotective effects of pink1 and LGR may be significant and useful in drug development.

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

    Directory of Open Access Journals (Sweden)

    W. Romero-Fernandez

    2014-07-01

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

  6. Tyrosine phosphorylation of Grb14 by Tie2

    Directory of Open Access Journals (Sweden)

    Dumont Daniel J

    2010-10-01

    Full Text Available Abstract Background Growth factor receptor bound (Grb proteins 7, 10 and 14 are a family of structurally related multi-domain adaptor proteins involved in a variety of biological processes. Grb7, 10 and 14 are known to become serine and/or threonine phosphorylated in response to growth factor (GF stimulation. Grb7 and 10 have also been shown to become tyrosine phosphorylated under certain conditions. Under experimental conditions Grb7 is tyrosine phosphorylated by the Tie2/Tie-2/Tek angiogenic receptor tyrosine kinase (RTK. Furthermore, Grb14 has also been shown to interact with Tie2, however tyrosine phosphorylation of this Grb family member has yet to be reported. Results Here we report for the first time tyrosine phosphorylation of Grb14. This phosphorylation requires a kinase competent Tie2 as well as intact tyrosines 1100 and 1106 (Y1100 and Y1106 on the receptor. Furthermore, a complete SH2 domain on Grb14 is required for Grb14 tyrosine phosphorylation by Tie2. Grb14 was also able to become tyrosine phosphorylated in primary endothelial cells when treated with a soluble and potent variant of the Tie2 ligand, cartilage oligomeric matrix protein (COMP Ang1. Conclusion Our results show that Grb14, like its family members Grb7 and Grb10, is able to be tyrosine phosphorylated. Furthermore, our data indicate a role for Grb14 in endothelial signaling downstream of the Tie2 receptor.

  7. An artificial TCA cycle selects for efficient α-ketoglutarate dependent hydroxylase catalysis in engineered Escherichia coli.

    Science.gov (United States)

    Theodosiou, Eleni; Breisch, Marina; Julsing, Mattijs K; Falcioni, Francesco; Bühler, Bruno; Schmid, Andreas

    2017-07-01

    Amino acid hydroxylases depend directly on the cellular TCA cycle via their cosubstrate α-ketoglutarate (α-KG) and are highly useful for the selective biocatalytic oxyfunctionalization of amino acids. This study evaluates TCA cycle engineering strategies to force and increase α-KG flux through proline-4-hydroxylase (P4H). The genes sucA (α-KG dehydrogenase E1 subunit) and sucC (succinyl-CoA synthetase β subunit) were alternately deleted together with aceA (isocitrate lyase) in proline degradation-deficient Escherichia coli strains (ΔputA) expressing the p4h gene. Whereas, the ΔsucCΔaceAΔputA strain grew in minimal medium in the absence of P4H, relying on the activity of fumarate reductase, growth of the ΔsucAΔaceAΔputA strictly depended on P4H activity, thus coupling growth to proline hydroxylation. P4H restored growth, even when proline was not externally added. However, the reduced succinyl-CoA pool caused a 27% decrease of the average cell size compared to the wildtype strain. Medium supplementation partially restored the morphology and, in some cases, enhanced proline hydroxylation activity. The specific proline hydroxylation rate doubled when putP, encoding the Na + /l-proline transporter, was overexpressed in the ΔsucAΔaceAΔputA strain. This is in contrast to wildtype and ΔputA single-knock out strains, in which α-KG availability obviously limited proline hydroxylation. Such α-KG limitation was relieved in the ΔsucAΔaceAΔputA strain. Furthermore, the ΔsucAΔaceAΔputA strain was used to demonstrate an agar plate-based method for the identification and selection of active α-KG dependent hydroxylases. This together with the possibility to waive selection pressure and overcome α-KG limitation in respective hydroxylation processes based on living cells emphasizes the potential of TCA cycle engineering for the productive application of α-KG dependent hydroxylases. Biotechnol. Bioeng. 2017;114: 1511-1520. © 2017 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    Yu, Bingwu; Ruppert, Martin; Stöckigt, Joachim

    2002-08-01

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

  9. 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

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

    International Nuclear Information System (INIS)

    Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

    2013-01-01

    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

  11. 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.

  12. Free radical-mediated stimulation of tyrosine-specific protein kinase in rat liver plasma membrane

    International Nuclear Information System (INIS)

    Chan, T.M.; Tatoyan, A.; Cheng, E.; Shargill, N.S.; Pleta, M.

    1986-01-01

    Incorporation of 32 P from (γ- 32 P)-ATP into endogenous proteins of plasma membranes isolated from rat liver was significantly increased by several naphthoquinones including menadione. This apparent stimulation of membrane-associated protein kinase activity by these compounds was most striking (up to 6-7 fold) when the synthetic copolymers containing glutamate and tyrosine residues (4:1) was used as substrate. Since tyrosine residues are the only possible phosphate acceptor in the copolymers, the quinone-stimulated liver membrane protein kinase is most likely tyrosine specific. Although not required for protein kinase activity, dithiothreitol (DTT) was necessary for its stimulation by these quinonoid compounds. Hydrolysis of ATP was not significantly affected by quinones under the experimental conditions. Both menadione and vitamin k 5 increased phosphorylation of plasma membrane proteins of molecular weight 45 and 60 kd. The stimulatory effect of menadione on protein phosphorylation was prevented by the addition of superoxide dismutase. Dihydroxyfumerate, which spontaneously produces various radical species, and H 2 O 2 , also stimulated tyrosine-specific protein phosphorylation. DTT was also required for their full effect. It, therefore, appears that quinonone stimulation of tyrosine-specific protein phosphorylation is mediated by oxygen radicals

  13. Reduction in total plasma ghrelin levels following catecholamine depletion: relation to bulimic and depressive symptoms.

    Science.gov (United States)

    Homan, Philipp; Grob, Simona; Milos, Gabriella; Schnyder, Ulrich; Hasler, Gregor

    2013-09-01

    There is increasing preclinical and clinical evidence of the important role played by the gastric peptide hormone ghrelin in the pathogenesis of symptoms of depression and eating disorders. To investigate the role of ghrelin and its considered counterpart, peptide tyrosine tyrosine (PYY), in the development of bulimic and depressive symptoms induced by catecholamine depletion, we administered the tyrosine hydroxylase inhibitor alpha-methyl-paratyrosine (AMPT) in a randomized, double-blind, placebo-controlled crossover, single-site experimental trial to 29 healthy controls and 20 subjects with fully recovered bulimia nervosa (rBN). We found a decrease between preprandial and postprandial plasma ghrelin levels (psymptoms (psymptoms induced by catecholamine depletion. These findings suggest a relationship between catecholamines and ghrelin with depressive symptoms. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Raman scattering tensors of tyrosine.

    Science.gov (United States)

    Tsuboi, M; Ezaki, Y; Aida, M; Suzuki, M; Yimit, A; Ushizawa, K; Ueda, T

    1998-01-01

    Polarized Raman scattering measurements have been made of a single crystal of L-tyrosine by the use of a Raman microscope with the 488.0-nm exciting beam from an argon ion laser. The L-tyrosine crystal belongs to the space group P2(1)2(1)2(1) (orthorhombic), and Raman scattering intensities corresponding to the aa, bb, cc, ab and ac components of the crystal Raman tensor have been determined for each prominent Raman band. A similar set of measurements has been made of L-tyrosine-d4, in which four hydrogen atoms on the benzene ring are replaced by deuterium atoms. The effects of NH3-->ND3 and OH-->OD on the Raman spectrum have also been examined. In addition, depolarization ratios of some bands of L-tyrosine in aqueous solutions of pH 13 and pH 1 were examined. For comparison with these experimental results, on the other hand, ab initio molecular orbital calculations have been made of the normal modes of vibration and their associated polarizability oscillations of the L-tyrosine molecule. On the basis of these experimental data and by referring to the results of the calculations, discussions have been presented on the Raman tensors associated to some Raman bands, including those at 829 cm-1 (benzene ring breathing), 642 cm-1 (benzene ring deformation), and 432 cm-1 (C alpha-C beta-C gamma bending).

  15. Phytanoyl-CoA hydroxylase activity is induced by phytanic acid

    NARCIS (Netherlands)

    Zomer, A. W.; Jansen, G. A.; van der Burg, B.; Verhoeven, N. M.; Jakobs, C.; van der Saag, P. T.; Wanders, R. J.; Poll-The, B. T.

    2000-01-01

    Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a branched-chain fatty acid present in various dietary products such as milk, cheese and fish. In patients with Refsum disease, accumulation of phytanic acid occurs due to a deficiency of phytanoyl-CoA hydroxylase, a peroxisomal enzyme

  16. Dopamine and Mushroom Bodies in Drosophila: Experience-Dependent and -Independent Aspects of Sexual Behavior

    Science.gov (United States)

    Neckameyer, Wendi S.

    1998-01-01

    Depletion of dopamine in Drosophila melanogaster adult males, accomplished through systemic introduction of the tyrosine hydroxylase inhibitor 3-iodo-tyrosine, severely impaired the ability of these flies to modify their courtship responses to immature males. Mature males, when first exposed to immature males, will perform courtship rituals; the intensity and duration of this behavior rapidly diminshes with time. Dopamine is also required for normal female sexual receptivity; dopamine-depleted females show increased latency to copulation. One kilobase of 5′ upstream information from the Drosophila tyrosine hydroxylase (DTH) gene, when fused to the Escherichia coli β-galactosidase reporter and transduced into the genome of Drosophila melanogaster, is capable of directing expression of the reporter gene in the mushroom bodies, which are believed to mediate learning acquisition and memory retention in flies. Ablation of mushroom bodies by treatment of newly hatched larva with hydroxyurea resulted in the inability of treated mature adult males to cease courtship when placed with untreated immature males. However, functional mushroom bodies were not required for the dopaminergic modulation of an innate behavior, female sexual receptivity. These data suggest that dopamine acts as a signaling molecule within the mushroom bodies to mediate a simple form of learning. PMID:10454380

  17. Effects of excess dietary tyrosine or certain xenobiotics on the cholesterogenesis in rats

    International Nuclear Information System (INIS)

    Nagaoka, S.; Masaki, H.; Aoyama, Y.; Yoshida, A.

    1986-01-01

    Comparison of the effects of excess dietary tyrosine, DDT, chlorobutanol (Chloretone) or butylated hydroxyanisole (BHA) on serum cholesterol, hepatic activities of the rate-limiting enzyme of cholesterol synthesis,3-hydroxy-3-methylglutaryl coenzyme A reductase and in vivo rates of the hepatic cholesterol synthesis measured by 3 H 2 O incorporation were investigated in rats. Serum cholesterol concentration was significantly higher in rats fed the DDT, chlorobutanol, BHA or excess tyrosine diets than in rats fed the control diet for 7 days. Serum cholesterol concentration remained higher compared to control rats when excess tyrosine was fed for 21 d. When rats were fed a basal diet after feeding a tyrosine excess diet for 2 wk, liver weight and serum cholesterol level returned to normal within 7 d. The incorporation of 3 H 2 O into liver cholesterol and the activity of liver 3-hydroxy-3-methylglutaryl coenzyme A reductase were greater in rats fed excess tyrosine or certain xenobiotics than in control rats. Present results suggested that the increase in serum cholesterol concentration due to excess dietary tyrosine or certain xenobiotics is mainly attributable to the stimulation of liver cholesterol synthesis

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

    Science.gov (United States)

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

    2006-06-01

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

  19. Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer

    International Nuclear Information System (INIS)

    Rawluszko, Agnieszka A; Bujnicka, Katarzyna E; Horbacka, Karolina; Krokowicz, Piotr; Jagodziński, Paweł P

    2013-01-01

    Colorectal cancer (CRC) is one of the most common and comprehensively studied malignancies. Hypoxic conditions during formation of CRC may support the development of more aggressive cancers. Hypoxia inducible factor (HIF), a major player in cancerous tissue adaptation to hypoxia, is negatively regulated by the family of prolyl hydroxylase enzymes (PHD1, PHD2, PHD3) and asparaginyl hydroxylase, called factor inhibiting HIF (FIH). PHD1, PHD2, PHD3 and FIH gene expression was evaluated using quantitative RT-PCR and western blotting in primary colonic adenocarcinoma and adjacent histopathologically unchanged colonic mucosa from patients who underwent radical surgical resection of the colon (n = 90), and the same methods were used for assessment of PHD3 gene expression in HCT116 and DLD-1 CRC cell lines. DNA methylation levels of the CpG island in the promoter regulatory region of PHD1, PHD2, PHD3 and FIH were assessed using bisulfite DNA sequencing and high resolution melting analysis (HRM) for patients and HRM analysis for CRC cell lines. We found significantly lower levels of PHD1, PHD2 and PHD3 transcripts (p = 0.00026; p < 0.00001; p < 0.00001) and proteins (p = 0.004164; p = 0.0071; p < 0.00001) in primary cancerous than in histopathologically unchanged tissues. Despite this, we did not observe statistically significant differences in FIH transcript levels between cancerous and histopathologically unchanged colorectal tissue, but we found a significantly increased level of FIH protein in CRC (p = 0.0169). The reduced PHD3 expression was correlated with significantly increased DNA methylation in the CpG island of the PHD3 promoter regulatory region (p < 0.0001). We did not observe DNA methylation in the CpG island of the PHD1, PHD2 or FIH promoter in cancerous and histopathologically unchanged colorectal tissue. We also showed that 5-Aza-2’-deoxycytidine induced DNA demethylation leading to increased PHD3 transcript and protein level in HCT116 cells. We

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

    Science.gov (United States)

    Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

    2013-01-01

    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. PMID:23566956

  1. Phosphorylated c-Mpl tyrosine 591 regulates thrombopoietin-induced signaling.

    Science.gov (United States)

    Sangkhae, Veena; Saur, Sebastian Jonas; Kaushansky, Alexis; Kaushansky, Kenneth; Hitchcock, Ian Stuart

    2014-06-01

    Thrombopoietin (TPO) is the primary regulator of platelet production, affecting cell survival, proliferation, and differentiation through binding to and stimulation of the cell surface receptor the cellular myeloproliferative leukemia virus oncogene (c-Mpl). Activating mutations in c-Mpl constitutively stimulate downstream signaling pathways, leading to aberrant hematopoiesis, and contribute to development of myeloproliferative neoplasms. Several studies have mapped the tyrosine residues within the cytoplasmic domain of c-Mpl that mediate these cellular signals; however, secondary signaling pathways are incompletely understood. In this study, we focused on c-Mpl tyrosine 591 (Y591). We found Y591 of wild-type c-Mpl to be phosphorylated in the presence of TPO. Additionally, eliminating Y591 phosphorylation by mutation to Phe resulted in decreased total receptor phosphorylation. Using a Src homology 2/phosphotyrosine-binding (SH2/PTB) domain binding microarray, we identified novel c-Mpl binding partners for phosphorylated Y591, including Src homology region 2 domain-containing phosphatase-1 (SHP-1), spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK). The functional significance of binding partners was determined through small interfering RNA treatment of Ba/F3-Mpl cells, confirming that the increase in pERK1/2 resulting from removal of Y591 may be mediated by spleen tyrosine kinase. These findings identify a novel negative regulatory pathway that controls TPO-mediated signaling, advancing our understanding of the mechanisms required for successful maintenance of hematopoietic stem cells and megakaryocyte development. Copyright © 2014 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  2. 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

  3. Chronically reinforced, operant olfactory conditioning increases the number of newborn GABAergic olfactory periglomerular neurons in the adult rat.

    Science.gov (United States)

    Tapia-Rodríguez, Miguel; Esquivelzeta-Rabell, José F; Gutiérrez-Ospina, Gabriel

    2012-12-01

    The mammalian brain preserves the ability to replace olfactory periglomerular cells (PGC) throughout life. Even though we have detailed a great deal the mechanisms underlying stem and amplifying cells maintenance and proliferation, as well as those modulating migration and differentiation, our knowledge on PGC phenotypic plasticity is at best fragmented and controversial. Here we explored whether chronically reinforced olfactory conditioning influences the phenotype of newborn PGC. Accordingly, olfactory conditioned rats showed increased numbers of GAD 65/67 positive PGC. Because such phenotypic change was not accompanied neither by increments in the total number of PGC, or periglomerular cell nuclei labeled with bromodeoxyuridine, nor by reductions in the number of tyrosine hydroxylase (TH), calbindin (CB) or calretinin (CR) immunoreactive PGC, we speculate that increments in the number of GABAergic PGC occur at the expense of other PGC phenotypes. In any event, these results support that adult newborn PGC phenotype may be subjected to phenotypic plasticity influenced by sensory stimulation. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. 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

  5. Eosin-sensitized photooxidation of substituted phenylalanines and tyrosines

    Energy Technology Data Exchange (ETDEWEB)

    Rizzuto, F.; Spikes, J.D.

    1977-01-01

    The cosin-sensitized photooxidation of tyrosine and a number of compounds related to tyrosine (substituted phenylalanines) was studied by steady-state kinetic and flash photolysis techniques. In particular, the role of the phenolic group and the amino and carboxyl groups of the alanyl side chain in the photooxidation mechanism was investigated in detail. Several relationships between substrate structure and susceptibility to photooxidation as well as effects of substrate structure on photooxidation mechanisms were found. For example, phenylalanine is not photooxidizable, but substitution of electron-donating (activating) groups such as -OH (as in tyrosine) or -NH/sub 2/ (as in p-aminophenylalanine) results in rapidly photooxidized derivatives. However, substituting deactivating groups such as -Cl (as in p-chlorophenylalanine) or weakly activating groups such as -OCH/sub 3/ (as in 4-methoxyphenylalanine) result in non-photooxidizable derivatives. Substitution of additional activating groups to the ring of hydroxy-substituted phenylalanines results in increased rates of photooxidation, whereas additional deactivating groups result in decreased photooxidation rates. The rate-determining step in the photooxidation mechanism is shown to be dependent on the presence and position of an electron-donating substituent on the benzenoid ring. Only minor involvement of the side chain amino and carboxyl groups was found. Both singlet oxygen and hydrogen abstraction mechanisms are involved in the eosin-sensitized photooxidation of hydroxy-substituted phenylalanines (e.g., tyrosine). The hydrogen abstraction mechanism probably predominates at both pH 8 and 11.

  6. 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

  7. The dopamine beta-hydroxylase inhibitor nepicastat increases dopamine release and potentiates psychostimulant-induced dopamine release in the prefrontal cortex.

    Science.gov (United States)

    Devoto, Paola; Flore, Giovanna; Saba, Pierluigi; Bini, Valentina; Gessa, Gian Luigi

    2014-07-01

    The dopamine-beta-hydroxylase inhibitor nepicastat has been shown to reproduce disulfiram ability to suppress the reinstatement of cocaine seeking after extinction in rats. To clarify its mechanism of action, we examined the effect of nepicastat, given alone or in association with cocaine or amphetamine, on catecholamine release in the medial prefrontal cortex and the nucleus accumbens, two key regions involved in the reinforcing and motivational effects of cocaine and in the reinstatement of cocaine seeking. Nepicastat effect on catecholamines was evaluated by microdialysis in freely moving rats. Nepicastat reduced noradrenaline release both in the medial prefrontal cortex and in the nucleus accumbens, and increased dopamine release in the medial prefrontal cortex but not in the nucleus accumbens. Moreover, nepicastat markedly potentiated cocaine- and amphetamine-induced extracellular dopamine accumulation in the medial prefrontal cortex but not in the nucleus accumbens. Extracellular dopamine accumulation produced by nepicastat alone or by its combination with cocaine or amphetamine was suppressed by the α2 -adrenoceptor agonist clonidine. It is suggested that nepicastat, by suppressing noradrenaline synthesis and release, eliminated the α2 -adrenoceptor mediated inhibitory mechanism that constrains dopamine release and cocaine- and amphetamine-induced dopamine release from noradrenaline or dopamine terminals in the medial prefrontal cortex. © 2012 The Authors, Addiction Biology © 2012 Society for the Study of Addiction.

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

    OpenAIRE

    Alonso, Hernan; Roujeinikova, Anna

    2012-01-01

    The alkane hydroxylase system of Pseudomonas putida GPo1 allows it to use alkanes as the sole source of carbon and energy. Bacterial alkane hydroxylases have tremendous potential as biocatalysts for the stereo- and regioselective transformation of a wide range of chemically inert unreactive alkanes into valuable reactive chemical precursors. We have produced and characterized the first 2-dimensional crystals of the integral membrane component of the P. putida alkane hydroxylase system, the no...

  9. Importance of tyrosine phosphorylation in receptor kinase complexes.

    Science.gov (United States)

    Macho, Alberto P; Lozano-Durán, Rosa; Zipfel, Cyril

    2015-05-01

    Tyrosine phosphorylation is an important post-translational modification that is known to regulate receptor kinase (RK)-mediated signaling in animals. Plant RKs are annotated as serine/threonine kinases, but recent work has revealed that tyrosine phosphorylation is also crucial for the activation of RK-mediated signaling in plants. These initial observations have paved the way for subsequent detailed studies on the mechanism of activation of plant RKs and the biological relevance of tyrosine phosphorylation for plant growth and immunity. In this Opinion article we review recent reports on the contribution of RK tyrosine phosphorylation in plant growth and immunity; we propose that tyrosine phosphorylation plays a major regulatory role in the initiation and transduction of RK-mediated signaling in plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    Yang, H; Raizada, M K

    1998-09-01

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

  11. Conformational Structure of Tyrosine, Tyrosyl-Glycine, and Tyrosyl-Glycyl-Glycine by Double Resonance Spectroscopy

    Science.gov (United States)

    Abo-Riziq, Ali; Grace, Louis; Crews, Bridgit; Callahan, Michael P,; van Mourik, Tanja; de Vries, Mattanjah S,

    2011-01-01

    We investigated the variation in conformation for the amino acid tyrosine (Y), alone and in the small peptides tyrosine-glycine (YC) and tyrosine-glycine-glycine (YGG), in the gas phase by using UV-UV and IR-UV double resonance spectroscopy and density functional theory calculations. For tyrosine we found seven different conformations, for YG we found four different conformations, and for YGG we found three different conformations. As the peptides get larger, we observe fewer stable conformers, despite the increasing complexity and number of degrees of freedom. We find structural trends similar to those in phenylalanine-glycine glycine (FGG) and tryptophan-glycine-glycine (WGG)j however) the effect of dispersive forces in FGG for stabilizing a folded structure is replaced by that of hydrogen bonding in YGG.

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

    Science.gov (United States)

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

    2000-06-01

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

  13. Phosphotyrosine phosphatase and tyrosine kinase inhibition modulate airway pressure-induced lung injury.

    Science.gov (United States)

    Parker, J C; Ivey, C L; Tucker, A

    1998-11-01

    We determined whether drugs which modulate the state of protein tyrosine phosphorylation could alter the threshold for high airway pressure-induced microvascular injury in isolated perfused rat lungs. Lungs were ventilated for successive 30-min periods with peak inflation pressures (PIP) of 7, 20, 30, and 35 cmH2O followed by measurement of the capillary filtration coefficient (Kfc), a sensitive index of hydraulic conductance. In untreated control lungs, Kfc increased by 1.3- and 3.3-fold relative to baseline (7 cmH2O PIP) after ventilation with 30 and 35 cmH2O PIP. However, in lungs treated with 100 microM phenylarsine oxide (a phosphotyrosine phosphatase inhibitor), Kfc increased by 4.7- and 16.4-fold relative to baseline at these PIP values. In lungs treated with 50 microM genistein (a tyrosine kinase inhibitor), Kfc increased significantly only at 35 cmH2O PIP, and the three groups were significantly different from each other. Thus phosphotyrosine phosphatase inhibition increased the susceptibility of rat lungs to high-PIP injury, and tyrosine kinase inhibition attenuated the injury relative to the high-PIP control lungs.

  14. 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.

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

    Science.gov (United States)

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

    2006-01-01

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

  16. Persistent expression of BMP-4 in embryonic chick adrenal cortical cells and its role in chromaffin cell development

    Directory of Open Access Journals (Sweden)

    Halbach Oliver

    2008-10-01

    Full Text Available Abstract Background Adrenal chromaffin cells and sympathetic neurons both originate from the neural crest, yet signals that trigger chromaffin development remain elusive. Bone morphogenetic proteins (BMPs emanating from the dorsal aorta are important signals for the induction of a sympathoadrenal catecholaminergic cell fate. Results We report here that BMP-4 is also expressed by adrenal cortical cells throughout chick embryonic development, suggesting a putative role in chromaffin cell development. Moreover, bone morphogenetic protein receptor IA is expressed by both cortical and chromaffin cells. Inhibiting BMP-4 with noggin prevents the increase in the number of tyrosine hydroxylase positive cells in adrenal explants without affecting cell proliferation. Hence, adrenal BMP-4 is likely to induce tyrosine hydroxylase in sympathoadrenal progenitors. To investigate whether persistent BMP-4 exposure is able to induce chromaffin traits in sympathetic ganglia, we locally grafted BMP-4 overexpressing cells next to sympathetic ganglia. Embryonic day 8 chick sympathetic ganglia, in addition to principal neurons, contain about 25% chromaffin-like cells. Ectopic BMP-4 did not increase this proportion, yet numbers and sizes of 'chromaffin' granules were significantly increased. Conclusion BMP-4 may serve to promote specific chromaffin traits, but is not sufficient to convert sympathetic neurons into a chromaffin phenotype.

  17. Enhancement of Naringenin Biosynthesis from Tyrosine by Metabolic Engineering of Saccharomyces cerevisiae.

    Science.gov (United States)

    Lyu, Xiaomei; Ng, Kuan Rei; Lee, Jie Lin; Mark, Rita; Chen, Wei Ning

    2017-08-09

    Flavonoids are an important class of plant polyphenols that possess a variety of health benefits. In this work, S. cerevisiae was metabolically engineered to produce the flavonoid naringenin, using tyrosine as the precursor. Our strategy to improve naringenin production comprised three modules. In module 1, we employed a modified GAL system to overexpress the genes of the naringenin biosynthesis pathway and investigated their synergistic action. In module 2, we simultaneously up-regulated acetyl-CoA production and down-regulated fatty acid biosynthesis in order to increase the precursor supply, malonyl-CoA. In module 3, we engineered the tyrosine biosynthetic pathway to eliminate the feedback inhibition of tyrosine and also down-regulated competing pathways. It was found that modules 1 and 3 played important roles in improving naringenin production. We succeeded in producing up to ∼90 mg/L of naringenin in our final strain, which is a 20-fold increase as compared to the parental strain.

  18. Increased protein expression of LHCG receptor and 17α-hydroxylase/17-20-lyase in human polycystic ovaries.

    Science.gov (United States)

    Comim, F V; Teerds, K; Hardy, K; Franks, S

    2013-11-01

    Does the expression of LHCG receptor (LHCGR) protein and key enzymes in the androgen biosynthetic pathway differ in normal human versus polycystic ovarian tissue? LHCGR and 17α-hydroxylase/17-20-lyase (CYP17A1) protein levels are increased in polycystic ovaries (PCOs). The predominant source of excess androgen secretion in women with polycystic ovary syndrome (PCOS) is ovarian theca cells but few studies have directly assessed the presence and abundance of protein for key molecules involved in androgen production by theca, including LHCGR and the rate-limiting enzyme in androgen production, CYP17A1. This is a laboratory-based, cross-sectional study comparing protein expression of key molecules in the androgen biosynthetic pathway in archived ovarian tissue from women with normal ovaries (n = 10) with those with PCOs (n = 16). A quantitative morphometric study was performed using sections of archived human ovaries (n = 26) previously characterized as normal or polycystic. The distribution and abundance of LHCGR, CYP17A1, 3β-hydroxysteroid dehydrogenase type 2 (3βHSDII) and 17β-hydroxysteroid dehydrogenase type 5 (17βHSD5) proteins were evaluated by immunohistochemistry and quantified. A higher proportion of theca cells from anovulatory PCO expressed LHCGR protein when compared with control ovaries (P = 0.01). A significant increase in the intensity of immunostaining for CYP17A1 was identified in antral follicles in sections of PCO compared with ovaries from normal women (P = 0.04). As the study used formalin-fixed ovarian tissue sections, it was not possible to carry out studies 'in vitro' using the same ovarian tissues in order to also demonstrate increased functional activity of LHCGR and CYP17A1. The data are in keeping with the results of previous studies in isolated theca cells and support the notion of an intrinsic abnormality of theca cell androgen production in women with PCOS. The research was supported by a Programme Grant, G0802782, from the Medical

  19. The metabolism of C14-labeled phenylalanine and tyrosine in malaria-infected Culex-females

    International Nuclear Information System (INIS)

    Maier, W.A.; Nassif-Makki, H.

    1975-01-01

    Culex females are fed on C14-phenylalanine or C14-tyrosine in sugar solution. Autoradiographic studies on homogenated females 1 or 4 days after feeding, show that the labeled amino acids are metabolized on the first day and are not detectable on the fourth day. After increase of the amino acid concentration by saturation of the sugar solution with the unlabeled amino acid, the labeled acid and its metabolites are visible over a longer period of time. Phenylalanine is metabolized to tyrosine and at least four other substances. Radioactivity on the starting point of the chromatogram can be interpreted as incorporation of tyrosine into proteins. After infection with Plasmodium cathemerium, and feeding of C14-phenylalanine C14-tyrosine is demonstrable over a longer period. (orig.) [de

  20. Synthesis of 14C-labelled α-methyl tyrosine

    International Nuclear Information System (INIS)

    Rajagopal, S.; Venkatachalam, T.K.; Conway, T.; Diksic, M.

    1992-01-01

    A new route for the preparation of radioactively labelled α-methyl L-tyrosine is described. The labelling at the α position has been successfully achieved with 14 C-, 11 C- (very preliminary, unpublished), and 3 H-labelled methyl iodide. A detailed report on 14 C-labelling at the α position and the hydrolysis of 4-methoxy α-methyl phenylalanine is presented. The alkylation proceeds via the methylation of the carbanion of N-benzylidene 4-methoxy phenylalanine methyl ester 2. Hydrolysis of 4-O methyl tyrosine to tyrosine by HBr and HI were analysed and used in the optimization of the hydrolysis conditions of 4. Enantiomeric purity of the isolated L-isomer has been found to be 99% as judged by HPLC. Pseudo first-order rate constant for the hydrolysis of 14 C-labelled α-methyl 4-methoxy phenyl alanine methyl ester was determined. Preliminary findings of the 3 H- and 11 C-radiolabelled α-methyl tyrosine (methyl labelled) are also mentioned. For the first time it was shown that α-methyl D,L-tyrosine can be separated into enantiomerically pure α-methyl D- and L-tyrosine using a CHIRALPAK WH column. (author)

  1. Novel vitamin D 1α-hydroxylase gene mutations in a Chinese ...

    Indian Academy of Sciences (India)

    2011-08-19

    Aug 19, 2011 ... known as vitamin D 1α-hydroxylase deficiency or pseu- dovitamin D ... amplicons of the 378 bp were digested with restriction enzyme PvuI and ... have no enzymatic activity; a missense mutation c.473T>C. (p.L158P) in the ...

  2. Oral l-tyrosine supplementation augments the vasoconstriction response to whole-body cooling in older adults.

    Science.gov (United States)

    Lang, James A; Smaller, Kevin A

    2017-07-01

    What is the central question of this study? Ageing is associated with altered sympathetic responses to stress, which are explained in part by reduced noradrenergic function. The impact of supplementation with oral l-tyrosine, the amino acid precursor for catecholamine synthesis, on the effector responses to cold and exercise stress has yet to be examined. What is the main finding and its importance? Oral l-tyrosine ingestion augmented the sympathetically mediated vasoconstriction response to cold exposure in aged skin. This suggests that l-tyrosine supplementation might improve thermoregulatory function in older adults. l-Tyrosine is the primary substrate for noradrenaline biosynthesis within sympathetic axon terminals. In stressful conditions requiring increased catecholamine production, the axonal l-tyrosine concentration may limit the full expression of the sympathetic effector response and this may be particularly evident in older adults. We hypothesize that oral l-tyrosine supplementation will increase the sympathetic response to whole-body cooling and muscle metaboreflex activation. In a randomized, double-blind design, 11 young (Y = 24 ± 1 years) and 11 older participants (O = 68 ± 4 years) ingested either 150 mg kg -1 of l-tyrosine or placebo before commencing 30 min of whole-body cooling to induce a gradual decline in skin temperature from 34 to 30.5°C. Laser Doppler flux (LDF) was measured at the ventral forearm, and cutaneous vascular conductance (CVC) was calculated as CVC = LDF/mean arterial pressure and expressed as a percentage change from baseline (%ΔCVC). Two minutes of static hand-grip exercise (35% maximal voluntary contraction) followed by 3 min of postexercise ischaemia were implemented before and toward the end of the cooling bout. l-Tyrosine supplementation did not affect blood pressure or heart rate responses to exercise or postexercise ischaemia. However, the blunted vasoconstriction response to whole-body cooling in

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

    Science.gov (United States)

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

    2013-01-01

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

  4. Tyrosine phosphorylation of WW proteins

    Science.gov (United States)

    Reuven, Nina; Shanzer, Matan

    2015-01-01

    A number of key regulatory proteins contain one or two copies of the WW domain known to mediate protein–protein interaction via proline-rich motifs, such as PPxY. The Hippo pathway components take advantage of this module to transduce tumor suppressor signaling. It is becoming evident that tyrosine phosphorylation is a critical regulator of the WW proteins. Here, we review the current knowledge on the involved tyrosine kinases and their roles in regulating the WW proteins. PMID:25627656

  5. Renal albumin excretion: twin studies identify influences of heredity, environment, and adrenergic pathway polymorphism

    DEFF Research Database (Denmark)

    Rao, Fangwen; Wessel, Jennifer; Wen, Gen

    2007-01-01

    biosynthesis (tyrosine hydroxylase), catabolism (monoamine oxidase A), storage/release (chromogranin A), receptor target (dopamine D1 receptor), and postreceptor signal transduction (sorting nexin 13 and rho kinase). Epistasis (gene-by-gene interaction) occurred between alleles at rho kinase, tyrosine...... hydroxylase, chromogranin A, and sorting nexin 13. Dopamine D1 receptor polymorphism showed pleiotropic effects on both albumin and dopamine excretion. These studies establish new roles for heredity and environment in albumin excretion. Urinary excretions of albumin and catecholamines are highly heritable......, and their parallel suggests adrenergic mediation of early glomerular permeability alterations. Albumin excretion is influenced by multiple adrenergic pathway genes and is, thus, polygenic. Such functional links between adrenergic activity and glomerular injury suggest novel approaches to its prediction, prevention...

  6. 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

  7. Tyrosine 402 Phosphorylation of Pyk2 Is Involved in Ionomycin-Induced Neurotransmitter Release

    Science.gov (United States)

    Zhang, Zhao; Zhang, Yun; Mou, Zheng; Chu, Shifeng; Chen, Xiaoyu; He, Wenbin; Guo, Xiaofeng; Yuan, Yuhe; Takahashi, Masami; Chen, Naihong

    2014-01-01

    Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca2+ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2), in clonal rat pheochromocytoma PC12 cells and cerebellar granule cells, whereas introduction of Pyk2 siRNA dramatically suppressed ionomycin-induced neurotransmitter release. Further study indicated that Tyr-402 (Y402) in Pyk2, instead of other tyrosine sites, underwent rapid phosphorylation after ionomycin induction in 1 min to 2 min. We demonstrated that the mutant of Pyk2 Y402 could abolish ionomycin-induced dopamine (DA) release by transfecting cells with recombinant Pyk2 and its mutants (Y402F, Y579F, Y580F, and Y881F). In addition, Src inhibition could prolong phosphorylation of Pyk2 Y402 and increase DA release. These findings suggested that Pyk2 was involved in ionomycin-induced neurotransmitter release through phosphorylation of Y402. PMID:24718602

  8. 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.

  9. Serum markers in alkaptonuria: simultaneous analysis of homogentisic acid, tyrosine and nitisinone by liquid chromatography tandem mass spectrometry.

    Science.gov (United States)

    Hughes, Andrew T; Milan, Anna M; Davison, Andrew S; Christensen, Peter; Ross, Gordon; Gallagher, James A; Dutton, John J; Ranganath, Lakshminarayan R

    2015-09-01

    Alkaptonuria is a rare debilitating autosomal recessive disorder of tyrosine metabolism, where deficiency of homogentisate 1,2-dioxygenase results in increased homogentisic acid. Homogentisic acid is deposited as an ochronotic pigment in connective tissues, especially cartilage, leading to a severe early onset form of osteoarthritis, increased renal and prostatic stone formation and hardening of heart vessels. Treatment with the orphan drug, nitisinone, an inhibitor of 4-hydroxyphenylpyruvate dioxygenase has been shown to reduce urinary excretion of homogentisic acid. A reverse phase liquid chromatography tandem mass spectrometry method has been developed to simultaneously analyse serum homogentisic acid, tyrosine and nitisinone. Using matrix-matched calibration standards, two product ion transitions were identified for each compound (homogentisic acid, tyrosine, nitisinone) and their respective isotopically labelled internal standards ((13)C6-homogentisic acid, d2-tyrosine, (13)C6-nitisinone). Intrabatch accuracy was 94-108% for homogentisic acid, 95-109% for tyrosine and 89-106% for nitisinone; interbatch accuracy (n = 20) was 88-108% for homogentisic acid, 91-104% for tyrosine and 88-103% for nitisinone. Precision, both intra- and interbatch were alkaptonuria patients, pre- and post-nitisinone therapy. © The Author(s) 2015.

  10. Rapid enzymatic analysis of plasma for tyrosine.

    Science.gov (United States)

    Shimizu, H; Taniguchi, K; Sugiyama, M; Kanno, T

    1990-01-01

    In this rapid, simple, and convenient enzymatic method for measurement of tyrosine in plasma, tyrosine is converted to tyramine by action of tyrosine decarboxylase (EC 4.1.1.25) and the tyramine produced is oxidized to p-hydroxybenzyl aldehyde and hydrogen peroxide by action of tyramine oxidase (EC 1.4.3.9). The hydrogen peroxide is reacted with 4-aminoantipyrine and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine in the presence of peroxidase (EC 1.11.1.7) to obtain quinoneimine dye, the absorbance of which is measured at 570 nm. Thus tyrosine is measured in the visible range. The CV was 4.6% or less, and the measurement was unaffected by other amino acids, except for phenylalanine. The values obtained (y) correlated well with those obtained with an amino acid analyzer (x): y = 0.902x + 3.92 mumol/L (Syx = 12.3; r = 0.985; n = 54).

  11. Acrolein acts as a neurotoxin in the nigrostriatal dopaminergic system of rat: involvement of ?-synuclein aggregation and programmed cell death

    OpenAIRE

    Wang, Yi-Ting; Lin, Hui-Ching; Zhao, Wei-Zhong; Huang, Hui-Ju; Lo, Yu-Li; Wang, Hsiang-Tsui; Maan-Yuh Lin, Anya

    2017-01-01

    Clinical studies report significant increases in acrolein (an ?,?-unsaturated aldehyde) in the substantia nigra (SN) of patients with Parkinson?s disease (PD). In the present study, acrolein-induced neurotoxicity in the nigrostriatal dopaminergic system was investigated by local infusion of acrolein (15, 50, 150?nmoles/0.5??l) in the SN of Sprague-Dawley rats. Acrolein-induced neurodegeneration of nigrostriatal dopaminergic system was delineated by reductions in tyrosine hydroxylase (TH) leve...

  12. 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

    Superoxide reacts rapidly with other radicals, but these reactions have received little attention in the context of oxidative stress. For tyrosyl radicals, reaction with superoxide is 3-fold faster than dimerization, and forms the addition product tyrosine hydroperoxide. We have explored structural...... 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...... formed stable hydroperoxides, but N-acetyltyrosine and p-hydroxyphenylacetic acid did not, demonstrating a requirement for a free amino group. Using [14C]tyrosine, the hydroperoxide and dityrosine were formed at a molar ratio of 1.8:1. Studies with pre-formed hydroperoxides, and measurements of substrate...

  13. Mechanism of Cytochrome P450 17A1-Catalyzed Hydroxylase and Lyase Reactions

    DEFF Research Database (Denmark)

    Bonomo, Silvia; Jorgensen, Flemming Steen; Olsen, Lars

    2017-01-01

    Cytochrome P450 17A1 (CYP17A1) catalyzes C17 hydroxylation of pregnenolone and progesterone and the subsequent C17–C20 bond cleavage (lyase reaction) to form androgen precursors. Compound I (Cpd I) and peroxo anion (POA) are the heme-reactive species underlying the two reactions. We have characte...... the concept that the selectivity of the steroidogenic CYPs is ruled by direct interactions with the enzyme, in contrast to the selectivity of drug-metabolizing CYPs, where the reactivity of the substrates dominates....... characterized the reaction path for both the hydroxylase and lyase reactions using density functional theory (DFT) calculations and the enzyme–substrate interactions by molecular dynamics (MD) simulations. Activation barriers for positions subject to hydroxylase reaction have values close to each other and span...

  14. Tyrosine Phosphorylation of the Human Serotonin Transporter: A Role in the Transporter Stability and Function

    Science.gov (United States)

    Annamalai, Balasubramaniam; Mannangatti, Padmanabhan; Arapulisamy, Obulakshmi; Shippenberg, Toni S.; Jayanthi, Lankupalle D.

    2012-01-01

    The serotonin (5-HT) transporter (SERT) regulates serotoninergic neurotransmission by clearing 5-HT released into the synaptic space. Phosphorylation of SERT on serine and threonine mediates SERT regulation. Whether tyrosine phosphorylation regulates SERT is unknown. Here, we tested the hypothesis that tyrosine-phosphorylation of SERT regulates 5-HT transport. In support of this, alkali-resistant 32P-labeled SERT was found in rat platelets, and Src-tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4,d]pyrimidine (PP2) decreased platelet SERT function and expression. In human placental trophoblast cells expressing SERT, PP2 reduced transporter function, expression, and stability. Although siRNA silencing of Src expression decreased SERT function and expression, coexpression of Src resulted in PP2-sensitive increases in SERT function and expression. PP2 treatment markedly decreased SERT protein stability. Compared with WT-SERT, SERT tyrosine mutants Y47F and Y142F exhibited reduced 5-HT transport despite their higher total and cell surface expression levels. Moreover, Src-coexpression increased total and cell surface expression of Y47F and Y142F SERT mutants without affecting their 5-HT transport capacity. It is noteworthy that Y47F and Y142F mutants exhibited higher protein stability compared with WT-SERT. However, similar to WT-SERT, PP2 treatment decreased the stability of Y47F and Y142F mutants. Furthermore, compared with WT-SERT, Y47F and Y142F mutants exhibited lower basal tyrosine phosphorylation and no further enhancement of tyrosine phosphorylation in response to Src coexpression. These results provide the first evidence that SERT tyrosine phosphorylation supports transporter protein stability and 5HT transport. PMID:21992875

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

    Science.gov (United States)

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

    2017-09-01

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

  16. Insulin growth factors regulate the mitotic cycle in cultured rat sympathetic neuroblasts

    International Nuclear Information System (INIS)

    DiCicco-Bloom, E.; Black, I.B.

    1988-01-01

    While neuronal mitosis is uniquely restricted to early development, the underlying regulation remains to be defined. The authors have now developed a dissociated, embryonic sympathetic neuron culture system that uses fully defined medium in which cells enter the mitotic cycle. The cultured cells expressed two neuronal traits, tyrosine hydroxylase and the neuron-specific 160-kDa neurofilament subunit protein, but were devoid of glial fibrillary acidic protein, a marker for non-myelin-forming Schwann cells in ganglia. Approximately one-third of the tyrosine hydroxylase-positive cells synthesized DNA in culture, specifically incorporating [ 3 H]thymidine into their nuclei. They used this system to define factors regulating the mitotic cycle in sympathetic neuroblasts. Members of the insulin family of growth factors, including insulin and insulin-like growth factors I and II, regulated DNA synthesis in the presumptive neuroblasts. Insulin more than doubled the proportion of tyrosine hydroxylase-positive cells entering the mitotic cycle, as indicated by autoradiography of [ 3 H]thymidine incorporation into nuclei. Scintillation spectrometry was an even more sensitive index of DNA synthesis. In contrast, the trophic protein nerve growth factor exhibited no mitogenic effect, suggesting that the mitogenic action of insulin growth factors is highly specific. The observations are discussed in the context of the detection of insulin growth factors and receptors in the developing brain

  17. Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

    Science.gov (United States)

    Chung, Heesung; Jung, Hyejung; Lee, Jung-Hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

    2014-08-01

    Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Science.gov (United States)

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

    2010-03-10

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

  19. Tyrosine Residues Regulate Multiple Nuclear Functions of P54nrb.

    Science.gov (United States)

    Lee, Ahn R; Hung, Wayne; Xie, Ning; Liu, Liangliang; He, Leye; Dong, Xuesen

    2017-04-01

    The non-POU-domain-containing octamer binding protein (NONO; also known as p54nrb) has various nuclear functions ranging from transcription, RNA splicing, DNA synthesis and repair. Although tyrosine phosphorylation has been proposed to account for the multi-functional properties of p54nrb, direct evidence on p54nrb as a phosphotyrosine protein remains unclear. To investigate the tyrosine phosphorylation status of p54nrb, we performed site-directed mutagenesis on the five tyrosine residues of p54nrb, replacing the tyrosine residues with phenylalanine or alanine, and immunoblotted for tyrosine phosphorylation. We then preceded with luciferase reporter assays, RNA splicing minigene assays, co-immunoprecipitation, and confocal microscopy to study the function of p54nrb tyrosine residues on transcription, RNA splicing, protein-protein interaction, and cellular localization. We found that p54nrb was not phosphorylated at tyrosine residues. Rather, it has non-specific binding affinity to anti-phosphotyrosine antibodies. However, replacement of tyrosine with phenylalanine altered p54nrb activities in transcription co-repression and RNA splicing in gene context-dependent fashions by means of differential regulation of p54nrb protein association with its interacting partners and co-regulators of transcription and splicing. These results demonstrate that tyrosine residues, regardless of phosphorylation status, are important for p54nrb function. J. Cell. Physiol. 232: 852-861, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  20. A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases.

    Science.gov (United States)

    Shen, S H; Bastien, L; Posner, B I; Chrétien, P

    1991-08-22

    The phosphorylation of proteins at tyrosine residues is critical in cellular signal transduction, neoplastic transformation and control of the mitotic cycle. These mechanisms are regulated by the activities of both protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPases). As in the PTKs, there are two classes of PTPases: membrane associated, receptor-like enzymes and soluble proteins. Here we report the isolation of a complementary DNA clone encoding a new form of soluble PTPase, PTP1C. The enzyme possesses a large noncatalytic region at the N terminus which unexpectedly contains two adjacent copies of the Src homology region 2 (the SH2 domain) found in various nonreceptor PTKs and other cytoplasmic signalling proteins. As with other SH2 sequences, the SH2 domains of PTP1C formed high-affinity complexes with the activated epidermal growth factor receptor and other phosphotyrosine-containing proteins. These results suggest that the SH2 regions in PTP1C may interact with other cellular components to modulate its own phosphatase activity against interacting substrates. PTPase activity may thus directly link growth factor receptors and other signalling proteins through protein-tyrosine phosphorylation.

  1. Effects of methyl jasmonate, on stevioside and rebaudioside A content and expression of the ent-Kaurenoic acid 13-hydroxylase gene in Stevia rebaudiana Bert. in vitro

    Directory of Open Access Journals (Sweden)

    Mehrdad Behmanesh

    2014-08-01

    Full Text Available Glycosides are a form of secondary metabolites that consist variety compounds and in some cases can play a role in primary metabolism. Steviol is lipophilic skeleton of Stevioside and Rebaudioside A, two main glycosides of Stevia rebuadiana. Steviol glycosides which are synthesized in S.rebaudiana have important medical and nutritional values as high intensity natural sweeteners. Steviol is synthesized from Kaurenoic acid in chloroplastic Terpenoid pathway that mediated by Kaurenoic acid 13-hydroxylase. In this study, HPLC method and RT-PCR were performed for quantification of glycosides and gene expression (ent-Kaurenoic acid 13-hydroxylase respectively. Methyl jasmonate treatment (at 20 micromolar in vitro induced glycoside biosynthesis significantly (P≤0.05 whereas higher concentration of Methyl jasmonate (100 µM caused a decrease in glycoside production and growth. The most glycoside content of the plant was three days after treatment. Also Methyl jasmonate treatment caused an increase in ent-Kaurenoic 13-hydroxylase gene expression from 6 hours to 48 hours (after treatment Results showed that biosynthesis of Stevia glycosides was probably a defense mechanism against pathogens and herbivore insects. Also we found that different concentrations of Methyl jasmonate, alter the ratio between glycosides rather than the increase in glycoside contents.

  2. ZDHHC3 Tyrosine Phosphorylation Regulates Neural Cell Adhesion Molecule Palmitoylation

    Science.gov (United States)

    Lievens, Patricia Marie-Jeanne; Kuznetsova, Tatiana; Kochlamazashvili, Gaga; Cesca, Fabrizia; Gorinski, Natalya; Galil, Dalia Abdel; Cherkas, Volodimir; Ronkina, Natalia; Lafera, Juri; Gaestel, Matthias

    2016-01-01

    The neural cell adhesion molecule (NCAM) mediates cell-cell and cell-matrix adhesion. It is broadly expressed in the nervous system and regulates neurite outgrowth, synaptogenesis, and synaptic plasticity. Previous in vitro studies revealed that palmitoylation of NCAM is required for fibroblast growth factor 2 (FGF2)-stimulated neurite outgrowth and identified the zinc finger DHHC (Asp-His-His-Cys)-containing proteins ZDHHC3 and ZDHHC7 as specific NCAM-palmitoylating enzymes. Here, we verified that FGF2 controlled NCAM palmitoylation in vivo and investigated molecular mechanisms regulating NCAM palmitoylation by ZDHHC3. Experiments with overexpression and pharmacological inhibition of FGF receptor (FGFR) and Src revealed that these kinases control tyrosine phosphorylation of ZDHHC3 and that ZDHHC3 is phosphorylated by endogenously expressed FGFR and Src proteins. By site-directed mutagenesis, we found that Tyr18 is an FGFR1-specific ZDHHC3 phosphorylation site, while Tyr295 and Tyr297 are specifically phosphorylated by Src kinase in cell-based and cell-free assays. Abrogation of tyrosine phosphorylation increased ZDHHC3 autopalmitoylation, enhanced interaction with NCAM, and upregulated NCAM palmitoylation. Expression of ZDHHC3 with tyrosine mutated in cultured hippocampal neurons promoted neurite outgrowth. Our findings for the first time highlight that FGFR- and Src-mediated tyrosine phosphorylation of ZDHHC3 modulates ZDHHC3 enzymatic activity and plays a role in neuronal morphogenesis. PMID:27247265

  3. Tyrosine-Nitrated Proteins: Proteomic and Bioanalytical Aspects.

    Science.gov (United States)

    Batthyány, Carlos; Bartesaghi, Silvina; Mastrogiovanni, Mauricio; Lima, Analía; Demicheli, Verónica; Radi, Rafael

    2017-03-01

    "Nitroproteomic" is under active development, as 3-nitrotyrosine in proteins constitutes a footprint left by the reactions of nitric oxide-derived oxidants that are usually associated to oxidative stress conditions. Moreover, protein tyrosine nitration can cause structural and functional changes, which may be of pathophysiological relevance for human disease conditions. Biological protein tyrosine nitration is a free radical process involving the intermediacy of tyrosyl radicals; in spite of being a nonenzymatic process, nitration is selectively directed toward a limited subset of tyrosine residues. Precise identification and quantitation of 3-nitrotyrosine in proteins has represented a "tour de force" for researchers. Recent Advances: A small number of proteins are preferential targets of nitration (usually less than 100 proteins per proteome), contrasting with the large number of proteins modified by other post-translational modifications such as phosphorylation, acetylation, and, notably, S-nitrosation. Proteomic approaches have revealed key features of tyrosine nitration both in vivo and in vitro, including selectivity, site specificity, and effects in protein structure and function. Identification of 3-nitrotyrosine-containing proteins and mapping nitrated residues is challenging, due to low abundance of this oxidative modification in biological samples and its unfriendly behavior in mass spectrometry (MS)-based technologies, that is, MALDI, electrospray ionization, and collision-induced dissociation. The use of (i) classical two-dimensional electrophoresis with immunochemical detection of nitrated proteins followed by protein ID by regular MS/MS in combination with (ii) immuno-enrichment of tyrosine-nitrated peptides and (iii) identification of nitrated peptides by a MIDAS™ experiment is arising as a potent methodology to unambiguously map and quantitate tyrosine-nitrated proteins in vivo. Antioxid. Redox Signal. 26, 313-328.

  4. 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

    Stimulation of GH receptors leads to rapid activation of Jak2 kinase and subsequent tyrosine phosphorylation of the GH receptor. Three specific tyrosines located in the C-terminal domain of the GH receptor have been identified as being involved in GH-stimulated transcription of the Spi 2.1 promoter....... 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......-binding activity, whereas the GH receptor mutant lacking all intracellular tyrosines was not. Synthetic tyrosine phosphorylated peptides corresponding to the GH receptor sequence around the three tyrosines inhibited Stat5 DNA-binding activity while their non-phosphorylated counterparts were ineffective. Tyrosine...

  5. Expression and purification of the metal-containing monooxygenases tryptophan hydroxylase and dopamine β-hydroxylase

    DEFF Research Database (Denmark)

    Karlsen, Pernille Efferbach

    -hyperactive disorder (ADHD) among others. Since all these diseases are the cause of huge economical and personal costs it is very important to gain more knowledge of TPH and DβH since these two enzymes could be possible targets for medicine against the diseases mentioned above. TPH a three-domain, iron......-containing enzyme which belongs to the aromatic amino acid hydroxylase (AAAH) family. It exist in two isoforms, TPH1 and TPH2, which are expressed in different tissues and have different properties. TPH is known as a very diffcult protein to work with especially due to instability and only truncated forms of TPH1...... have been purified and crystallized. This project concern the human neuronal TPH or TPH2. In an attempt to overcome the problems with recombinant TPH two stability and solubility optimized variants of TPH2 are designed. Escherichia coli (E. coli) expression strains for these variants and full length...

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

    Science.gov (United States)

    Ishiguro, Kanako; Taniguchi, Masumi; Tanaka, Yoshikazu

    2012-05-01

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

  7. Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium.

    Science.gov (United States)

    Murata, Yoji; Kotani, Takenori; Supriatna, Yana; Kitamura, Yasuaki; Imada, Shinya; Kawahara, Kohichi; Nishio, Miki; Daniwijaya, Edwin Widyanto; Sadakata, Hisanobu; Kusakari, Shinya; Mori, Munemasa; Kanazawa, Yoshitake; Saito, Yasuyuki; Okawa, Katsuya; Takeda-Morishita, Mariko; Okazawa, Hideki; Ohnishi, Hiroshi; Azuma, Takeshi; Suzuki, Akira; Matozaki, Takashi

    2015-08-04

    Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer-associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10-deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1-deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity.

  8. 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...... the high degree of functional and structural similarity between TC-PTP and PTP1B, we have been able to identify areas close to the active site that might be addressed to develop selective inhibitors of each enzyme....

  9. 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

  10. Differential in vivo regulation of TH and DBH mRNA in rat atria by maprotiline and fluoxetine

    Directory of Open Access Journals (Sweden)

    Spasojević Nataša

    2011-01-01

    Full Text Available It is well known that antidepressants affect central monoaminergic neurotransmission and that they also modulate hormone release in peripheral tissues. Repeated maprotiline (a noradrenaline reuptake inhibitor and fluoxetine (a serotonin reuptake inhibitor treatment on gene expression of the catecholamine biosynthetic enzymes were examined in rat atria and ventricles in vivo. Maprotiline decreased the gene expression of tyrosine hydroxylase (TH and dopamineβ-hydroxylase (DBH in the rat atrium. Fluoxetine increased gene expression of TH and DBH, but not of phenylethanolamine N-methyltransferase (PNMT. Chronic application of antidepressants did not change the expression of these enzymes in the ventricles. We conclude that repeated administration of fluoxetine enhances gene transcription of TH and DBH and subsequently stimulates noradrenaline synthesis in rat atria in vivo.

  11. Role of Mitochondrial DNA Mutations in Cellular Vulnerability to Mitochondria-Specific Environmental Toxins

    National Research Council Canada - National Science Library

    Hirsch, Etienne C

    2005-01-01

    .... To test such a hypothesis in Parkinson's disease we proposed to: 1) develop an animal model with accumulated mtDNA mutations in catecholaminergic neurons by creating a transgenic mouse containing a tyrosine hydroxylase (TH...

  12. Amelioration of behavioral abnormalities in BH(4-deficient mice by dietary supplementation of tyrosine.

    Directory of Open Access Journals (Sweden)

    Sang Su Kwak

    Full Text Available This study reports an amelioration of abnormal motor behaviors in tetrahydrobiopterin (BH4-deficient Spr (-/- mice by the dietary supplementation of tyrosine. Since BH4 is an essential cofactor for the conversion of phenylalanine into tyrosine as well as the synthesis of dopamine neurotransmitter within the central nervous system, the levels of tyrosine and dopamine were severely reduced in brains of BH4-deficient Spr (-/- mice. We found that Spr (-/- mice display variable 'open-field' behaviors, impaired motor functions on the 'rotating rod', and dystonic 'hind-limb clasping'. In this study, we report that these aberrant motor deficits displayed by Spr (-/- mice were ameliorated by the therapeutic tyrosine diet for 10 days. This study also suggests that dopamine deficiency in brains of Spr (-/- mice may not be the biological feature of aberrant motor behaviors associated with BH4 deficiency. Brain levels of dopamine (DA and its metabolites in Spr (-/- mice were not substantially increased by the dietary tyrosine therapy. However, we found that mTORC1 activity severely suppressed in brains of Spr (-/- mice fed a normal diet was restored 10 days after feeding the mice the tyrosine diet. The present study proposes that brain mTORC1 signaling pathway is one of the potential targets in understanding abnormal motor behaviors associated with BH4-deficiency.

  13. 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)

  14. Tetrahydrobiopterin precursor sepiapterin provides protection against neurotoxicity of 1-methyl-4-phenylpyridinium in nigral slice cultures

    DEFF Research Database (Denmark)

    Madsen, Jakob Torp; Jansen, Pernille; Hesslinger, Christian

    2003-01-01

    Complex-I inhibition and oxidative processes have been implicated in the loss of nigral dopamine neurones in Parkinson's disease and the toxicity of MPTP and its metabolite MPP+. Tetrahydrobiopterin, an essential cofactor for tyrosine hydroxylase, may act as an antioxidant in dopaminergic neurones...... and protects against the toxic consequences of glutathione depletion. Here we studied the effects of manipulating tetrahydrobiopterin levels on MPP+ toxicity in organotypic, rat ventral mesencephalic slice cultures. In cultures exposed to 30 micro m MPP+ for 2 days, followed by 8 days 'recovery' in control...... medium, we measured dopamine and its metabolites in the tissue and culture medium by HPLC, lactate dehydrogenase release to the culture medium, cellular uptake of propidium iodide and counted the tyrosine hydroxylase-immunoreactive neurones. Inhibition of tetrahydrobiopterin synthesis by 2,4-diamino-6...

  15. Putative tyrosine kinases expressed in K-562 human leukemia cells

    International Nuclear Information System (INIS)

    Partanen, J.; Maekelae, T.P.; Lehvaeslaiho, H.; Alitalo, K.; Alitalo, R.

    1990-01-01

    Tyrosine phosphorylation is important in the transmission of growth and differentiation signals; known tyrosine kinases include several oncoproteins and growth factor receptors. Interestingly, some differentiated cell types, such as erythrocytes and platelets contain high amounts of phosphotyrosine. The authors analyzed tyrosine kinases expressed in the K-562 chronic myelogenous leukemia cell line, which has a bipotential erythroid and megakaryoblastoid differentiation capacity. Analysis of 359 polymerase chain reaction-amplified cDNA clones led to the identification of 14 different tyrosine kinase-related sequences (JTK1-14). Two of the clones (JTK2 and JTK4) represent unusual members of the fibroblast growth factor receptor gene family, and the clones JTK5, JTK11, and JTK14 may also belong to the family of receptor tyrosine kinases but lack a close relationship to any known tyrosine kinase. Each of these different genes has its own characteristic expression pattern in K-562 cells and several other human tumor cell lines. In addition, the JTK11 and JTK14 mRNAs are induced during the megakaryoblastoid differentiation of K-562 cells. These tyrosine kinases may have a role in the differentiation of megakaryoblasts or in the physiology of platelets

  16. HFE Genotype Restricts the Response to Paraquat in a Mouse Model of Neurotoxicity.

    Science.gov (United States)

    Nixon, Anne M; Meadowcroft, Mark D; Neely, Elizabeth B; Snyder, Amanda M; Purnell, Carson J; Wright, Justin; Lamendella, Regina; Nandar, Wint; Huang, Xuemei; Connor, James R

    2018-05-01

    Parkinson's disease is marked clinically by motor dysfunction and pathologically by dopaminergic cell loss in the substantia nigra and iron accumulation in the substantia nigra. The driver underlying iron accumulation remains unknown and could be genetic or environmental. The HFE protein is critical for the regulation of cellular iron uptake. Mutations within this protein are associated with increased iron accumulation including in the brain. We have focused on the commonly occurring H63D variant of the HFE gene as a disease modifier in a number of neurodegenerative diseases. To investigate the role of H63D HFE genotype, we generated a mouse model in which the wild-type (WT) HFE gene is replaced by the H67D gene variant (mouse homolog of the human H63D gene variant). Using paraquat toxicity as the model for Parkinson's disease, we found that WT mice responded as expected with significantly greater motor function, loss of tyrosine hydroxylase staining and increase microglial staining in the substantia nigra, and an increase in R 2 relaxation rate within the substantia nigra of the paraquat-treated mice compared to their saline-treated counterparts. In contrast, the H67D mice showed a remarkable resistance to paraquat treatment; specifically differing from the WT mice with no changes in motor function or changes in R 2 relaxation rates following paraquat exposure. At baseline, there were differences between the H67D HFE mice and WT mice in gut microbiome profile and increased L-ferritin staining in the substantia nigra that could account for the resistance to paraquat. Of particular note, the H67D HFE mice regardless of whether or not they were treated with paraquat had significantly less tyrosine hydroxylase immunostaining than WT. Our results clearly demonstrate that the HFE genotype impacts the expression of tyrosine hydroxylase in the substantia nigra, the gut microbiome and the response to paraquat providing additional support that the HFE genotype is a disease

  17. 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...

  18. Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation

    DEFF Research Database (Denmark)

    Kirkegaard, Signe Skyum; Lambert, Ian Henry; Gammeltoft, Steen

    2010-01-01

    (K,vol) indicating that inhibition of RVD reflects inhibition of TASK-2. We find that in EATC the tyrosine kinase inhibitor genistein inhibits RVD by 90%, and that the tyrosine phosphatase inhibitor monoperoxo(picolinato)-oxo-vanadate(V) [mpV(pic)] shifted the volume set point for inactivation of the channel...... to a lower cell volume. Swelling-activated K(+) efflux was impaired by genistein and the Src kinase family inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and enhanced by the tyrosine phosphatase inhibitor mpV(pic). With the use of the TASK-2 inhibitor clofilium......, it is demonstrated that mpV(pic) increased the volume-sensitive part of the K(+) efflux 1.3 times. To exclude K(+) efflux via a KCl cotransporter, cellular Cl(-) was substituted with NO(3)(-). Also under these conditions K(+) efflux was completely blocked by genistein. Thus tyrosine kinases seem to be involved...

  19. Tyrosine kinase inhibitors: Multi-targeted or single-targeted?

    Science.gov (United States)

    Broekman, Fleur; Giovannetti, Elisa; Peters, Godefridus J

    2011-02-10

    Since in most tumors multiple signaling pathways are involved, many of the inhibitors in clinical development are designed to affect a wide range of targeted kinases. The most important tyrosine kinase families in the development of tyrosine kinase inhibitors are the ABL, SCR, platelet derived growth factor, vascular endothelial growth factor receptor and epidermal growth factor receptor families. Both multi-kinase inhibitors and single-kinase inhibitors have advantages and disadvantages, which are related to potential resistance mechanisms, pharmacokinetics, selectivity and tumor environment. In different malignancies various tyrosine kinases are mutated or overexpressed and several resistance mechanisms exist. Pharmacokinetics is influenced by interindividual differences and differs for two single targeted inhibitors or between patients treated by the same tyrosine kinase inhibitor. Different tyrosine kinase inhibitors have various mechanisms to achieve selectivity, while differences in gene expression exist between tumor and stromal cells. Considering these aspects, one type of inhibitor can generally not be preferred above the other, but will depend on the specific genetic constitution of the patient and the tumor, allowing personalized therapy. The most effective way of cancer treatment by using tyrosine kinase inhibitors is to consider each patient/tumor individually and to determine the strategy that specifically targets the consequences of altered (epi)genetics of the tumor. This strategy might result in treatment by a single multi kinase inhibitor for one patient, but in treatment by a couple of single kinase inhibitors for other patients.

  20. Interaction between O-GlcNAc modification and tyrosine phosphorylation of prohibitin: implication for a novel binary switch.

    Directory of Open Access Journals (Sweden)

    Sudharsana R Ande

    Full Text Available Prohibitin (PHB or PHB1 is an evolutionarily conserved, multifunctional protein which is present in various cellular compartments including the plasma membrane. However, mechanisms involved in various functions of PHB are not fully explored yet. Here we report for the first time that PHB interacts with O-linked beta-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT and is O-GlcNAc modified; and also undergoes tyrosine phosphorylation in response to insulin. Tyrosine 114 (Tyr114 and tyrosine 259 (Tyr259 in PHB are in the close proximity of potential O-GlcNAc sites serine 121 (Ser121 and threonine 258 (Thr258 respectively. Substitution of Tyr114 and Tyr259 residues in PHB with phenylalanine by site-directed mutagenesis results in reduced tyrosine phosphorylation as well as reduced O-GlcNAc modification of PHB. Surprisingly, this also resulted in enhanced tyrosine phosphorylation and activity of OGT. This is attributed to the presence of similar tyrosine motifs in PHB and OGT. Substitution of Ser121 and Thr258 with alanine and isoleucine respectively resulted in attenuation of O-GlcNAc modification and increased tyrosine phosphorylation of PHB suggesting an association between these two dynamic modifications. Sequence analysis of O-GlcNAc modified proteins having known O-GlcNAc modification site(s or known tyrosine phosphorylation site(s revealed a strong potential association between these two posttranslational modifications in various proteins. We speculate that O-GlcNAc modification and tyrosine phosphorylation of PHB play an important role in tyrosine kinase signaling pathways including insulin, growth factors and immune receptors signaling. In addition, we propose that O-GlcNAc modification and tyrosine phosphorylation is a novel previously unidentified binary switch which may provide new mechanistic insights into cell signaling pathways and is open for direct experimental examination.

  1. Measurement of protein synthesis: in vitro comparison of (68)Ga-DOTA-puromycin, [ (3)H]tyrosine, and 2-fluoro-[ (3)H]tyrosine.

    Science.gov (United States)

    Eigner, Sebastian; Beckford Vera, Denis R; Fellner, Marco; Loktionova, Natalia S; Piel, Markus; Melichar, Frantisek; Rösch, Frank; Roß, Tobias L; Lebeda, Ondrej; Henke, Katerina Eigner

    2013-01-01

    Puromycin has played an important role in our understanding of the eukaryotic ribosome and protein synthesis. It has been known for more than 40 years that this antibiotic is a universal protein synthesis inhibitor that acts as a structural analog of an aminoacyl-transfer RNA (aa-tRNA) in eukaryotic ribosomes. Due to the role of enzymes and their synthesis in situations of need (DNA damage, e.g., after chemo- or radiation therapy), determination of protein synthesis is important for control of antitumor therapy, to enhance long-term survival of tumor patients, and to minimize side-effects of therapy. Multiple attempts to reach this goal have been made through the last decades, mostly using radiolabeled amino acids, with limited or unsatisfactory success. The aim of this study is to estimate the possibility of determining protein synthesis ratios by using (68)Ga-DOTA-puromycin ((68)Ga-DOTA-Pur), [(3)H]tyrosine, and 2-fluoro-[(3)H]tyrosine and to estimate the possibility of different pathways due to the fluorination of tyrosine. DOTA-puromycin was synthesized using a puromycin-tethered controlled-pore glass (CPG) support by the usual protocol for automated DNA and RNA synthesis following our design. (68)Ga was obtained from a (68)Ge/(68)Ga generator as described previously by Zhernosekov et al. (J Nucl Med 48:1741-1748, 2007). The purified eluate was used for labeling of DOTA-puromycin at 95°C for 20 min. [(3)H]Tyrosine and 2-fluoro-[(3)H]tyrosine of the highest purity available were purchased from Moravek (Bera, USA) or Amersham Biosciences (Hammersmith, UK). In vitro uptake and protein incorporation as well as in vitro inhibition experiments using cycloheximide to inhibit protein synthesis were carried out for all three substances in DU145 prostate carcinoma cells (ATCC, USA). (68)Ga-DOTA-Pur was additionally used for μPET imaging of Walker carcinomas and AT1 tumors in rats. Dynamic scans were performed for 45 min after IV application (tail vein) of 20-25 MBq (68

  2. Hydroxyl radical induced cross-linking of cytosine and tyrosine in nucleohistone

    International Nuclear Information System (INIS)

    Gajewski, E.; Dizdaroglu, M.

    1990-01-01

    Hydroxyl radical induced formation of a DNA-protein cross-link involving cytosine and tyrosine in nucleohistone in buffered aqueous solution is reported. The technique of gas chromatography-mass spectrometry was used for this investigation. A γ-irradiated aqueous mixture of cytosine and tyrosine was first investigated in order to obtain gas chromatographic-mass spectrometric properties of possible cytosine-tyrosine cross-links. One cross-link was observed, and its structure was identified as the product from the formation of a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. With the use of gas chromatography-mass spectrometry with selected-ion monitoring, this cytosine-tyrosine cross-link was identified in acidic hydrolysates of calf thymus nucleohistone γ-irradiated in N 2 O-saturated aqueous solution. The yield of this DNA-protein cross-link in nucleohistone was found to be a linear function of the radiation dose in the range of 100-500 Gy (J·kg -1 ). This yield amounted to 0.05 nmol·J -1 . Mechanisms underlying the formation of the cytosine-tyrosine cross-link in nucleohistone were proposed to involve radical-radical and/or radical addition reactions of hydroxyl adduct radicals of cytosine and tyrosine moieties, forming a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. When oxygen was present in irradiated solutions, no cytosine-tyrosine cross-links were observed

  3. Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts

    DEFF Research Database (Denmark)

    Su, J; Muranjan, M; Sap, J

    1999-01-01

    of tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well...... established, no corresponding phosphatases have been identified that, under physiological conditions, function as positive regulators of c-Src and Fyn in fibroblasts. RESULTS: Receptor protein tyrosine phosphatase alpha (RPTPalpha) was inactivated by homologous recombination. Fibroblasts derived from...... these RPTPalpha-/- mice had impaired tyrosine kinase activity of both c-Src and Fyn, and this was accompanied by a concomitant increase in c-Src Tyr527 phosphorylation. RPTPalpha-/- fibroblasts also showed a reduction in the rate of spreading on fibronectin substrates, a trait that is a phenocopy of the effect...

  4. [Effects of perinatal exposure to bisphenol A inducing dopaminergic neuronal cell to apoptosis happening in midbrain of male rat offspring].

    Science.gov (United States)

    Lin, Yong; Zhang, Hao; Wang, Wen-dong; Wu, De-sheng; Jiang, Song-hui; Qu, Wei-dong

    2006-07-01

    To investigate the mechanism and effect of rat perinatal exposure to bisphenol A (BPA) resulting in midbrain dopaminergic neuronal cell apoptosis and tyrosine hydroxylase expression of male offspring. Rat dams were randomLy divided into 4 groups on gestational day(GD) 10 and given orally the bisphenol A doses as 0, 0.5, 5, 50 mg/kg x d from GD10 to weaning. The brains of male offspring were obtained for detecting, with immunohistochemistry protocol, the Caspase-3, Bcl-2 and tyrosine hydroxylase expression in the midbrain on postnatal day 21 or 30 respectively, and the midbrain apoptotic neuronal cell were detected by TUNEL on PND21. The expression of Caspase-3 in the midbrain of rat male offspring were increased but bcl-2 were decreased on PND21 and 30, respectively. On PND21, apoptotic neuronal cell were found in the midbrain of high and medium doses groups. TH protein expression was decreased. Perinatal exposure to bisphenol A can induce the apoptosis of midbrain dopaminergic neuron in the male rat offspring even after weaning, and concomitantly decrease the midbrain TH immunoreactivity, this may cause the abnormal function of dopaminergic pathway of rat male offspring.

  5. ROLE OF TYROSINE-SULFATED PROTEINS IN RETINAL STRUCTURE AND FUNCTION

    Science.gov (United States)

    Kanan, Y.; Al-Ubaidi, M.R.

    2014-01-01

    The extracellular matrix (ECM) plays a significant role in cellular and retinal health. The study of retinal tyrosine-sulfated proteins is an important first step toward understanding the role of ECM in retinal health and diseases. These secreted proteins are members of the retinal ECM. Tyrosine sulfation was shown to be necessary for the development of proper retinal structure and function. The importance of tyrosine sulfation is further demonstrated by the evolutionary presence of tyrosylprotein sulfotransferases, enzymes that catalyze proteins’ tyrosine sulfation, and the compensatory abilities of these enzymes. Research has identified four tyrosine-sulfated retinal proteins: fibulin 2, vitronectin, complement factor H (CFH), and opticin. Vitronectin and CFH regulate the activation of the complement system and are involved in the etiology of some cases of age-related macular degeneration. Analysis of the role of tyrosine sulfation in fibulin function showed that sulfation influences the protein's ability to regulate growth and migration. Although opticin was recently shown to exhibit anti-angiogenic properties, it is not yet determined what role sulfation plays in that function. Future studies focusing on identifying all of the tyrosine-sulfated retinal proteins would be instrumental in determining the impact of sulfation on retinal protein function in retinal homeostasis and diseases. PMID:25819460

  6. 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 ...

  7. Tyrosine phosphorylation in T cells is regulated by phosphatase activity: studies with phenylarsine oxide.

    OpenAIRE

    Garcia-Morales, P; Minami, Y; Luong, E; Klausner, R D; Samelson, L E

    1990-01-01

    Activation of T cells induces rapid tyrosine phosphorylation on the T-cell receptor zeta chain and other substrates. These phosphorylations can be regulated by a number of protein-tyrosine kinases (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). In this study, we demonstrate that phenylarsine oxide can inhibit tyrosine phosphatases while leaving tyrosine kinase function intact. We use this ...

  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. Robotic synthesis of L-[1-11C]tyrosine

    International Nuclear Information System (INIS)

    Luurtsema, Gert; Medema, Jitze; Elsinga, P.H.; Visser, G.M.; Vaalburg, Willem

    1994-01-01

    L-[1- 11 C]tyrosine promises to become an important tracer for determination of the protein synthesis rate (PSR) in tumor tissue and brain. The commercially available Anatech RB-86 robotic system is utilized for the automation of the L-[1- 11 C]tyrosine production via the isocyanide method as reported by Bolster et al. (Eur. J. Nucl. Med. 12, 321-324, 1986). The total synthesis time, including HPLC-purification and enantiomeric separation is 60 min. With a practical yield of 20 mCi L-[1- 11 C]tyrosine at a specific activity > 1000 Ci/mmol. (author)

  10. SUBSTRATE-SPECIFICITY OF THE ALKANE HYDROXYLASE SYSTEM OF PSEUDOMONAS-OLEOVORANS GPO1

    NARCIS (Netherlands)

    van Beilen, J.B.; Kingma, Jacob; Witholt, Bernard

    1994-01-01

    We have studied the hydroxylation of a wide range of linear, branched and cyclic alkanes and alkylbenzenes by the alkane hydroxylase system of Pseudomonas oleovorans GPo1 in vivo and in vitro. In vivo hydroxylation was determined with whole cells of the recombinant PpS8141; P. putida PpS81 carrying

  11. 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

  12. 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...

  13. Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by reverse-phase high-performance liquid chromatography using both endogenous and exogenous [4-14C]cholesterol as substrate

    International Nuclear Information System (INIS)

    Hylemon, P.B.; Studer, E.J.; Pandak, W.M.; Heuman, D.M.; Vlahcevic, Z.R.; Chiang, J.Y.

    1989-01-01

    The HPLC-spectrophotometric method for measuring cholesterol 7 alpha-hydroxylase activity was modified by using a C-18 reverse-phase column to separate 7 alpha-hydroxy-4-cholesten-3-one and 4-cholesten-3-one and by adding 7 beta-hydroxycholesterol to each reaction mixture as an internal recovery standard. With this method, we were able to simultaneously measure cholesterol 7 alpha-hydroxylase activity using endogenous cholesterol and exogenous [4- 14 C]cholesterol as substrate. Rat liver cytosol differentially stimulated (286%) the 7 alpha-hydroxylation of exogenous [4- 14 C]-cholesterol. In contrast, total cholesterol 7 alpha-hydroxylase activity was stimulated only 35% by cytosol. This method should prove useful for studying mechanisms of cholesterol delivery to cholesterol 7 alpha-hydroxylase

  14. 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.

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

    Science.gov (United States)

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

    2007-06-21

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

  16. Investigations into potential extrasynaptic communication between the dopaminergic and nitrergic systems

    Directory of Open Access Journals (Sweden)

    Miso eMitkovski

    2012-09-01

    Full Text Available Nitric oxide is unconstrained by cell membranes and can therefore act along a broad distance as a volume transmitter. Spillover of nitric oxide between neurons may have a major impact on central nervous system diseases and particularly on neurodegeneration. There is evidence whereby communication between nitrergic and dopaminergic systems plays an essential role in the control of the nigrostriatal pathway. However, there is sparse information for either the coexistence or overlap of nitric oxide and dopaminergic structures. The present study used double-labeling immunofluorescent microscopy to investigate the degree of cellular co-localization between nitric oxide synthase and tyrosine hydroxylase, enzymes responsible for the synthesis of nitric oxide and dopamine, respectively, was examined in neurons of the nigrostriatal pathway regions in the rat brain. After perfusional fixation, the brains were cut and double immunostained. A proximity analysis of tyrosine hydroxylase and nitric oxide synthase structures was made using confocal laser scanning microscopy, in nigrostriatal regions of the rat brain. We used image acquired at the optical limit and generated binary masks at 2µm-wide margin from the respective maximum projections. Co-localization between the two antigens was infrequent (<10% in most areas examined. However, tyrosine hydroxylase labeling was particularly concentrated close to nitric oxide synthase dendrites/axons and the cell bodies. These results further substantiate an extrasynaptic substrate for interaction between nitrergic and dopaminergic systems, thereby modulating sensitivity to neural inputs and its gene expression.

  17. Nigral dopaminergic neuron replenishment in adult mice through VE-cadherin-expressing neural progenitor cells

    Directory of Open Access Journals (Sweden)

    Abir A Rahman

    2017-01-01

    Full Text Available The function of dopaminergic neurons in the substantia nigra is of central importance to the coordination of movement by the brain's basal ganglia circuitry. This is evidenced by the loss of these neurons, resulting in the cardinal motor deficits associated with Parkinson's disease. In order to fully understand the physiology of these key neurons and develop potential therapies for their loss, it is essential to determine if and how dopaminergic neurons are replenished in the adult brain. Recent work has presented evidence for adult neurogenesis of these neurons by Nestin+/Sox2– neural progenitor cells. We sought to further validate this finding and explore a potential atypical origin for these progenitor cells. Since neural progenitor cells have a proximal association with the vasculature of the brain and subsets of endothelial cells are Nestin+, we hypothesized that dopaminergic neural progenitors might share a common cell lineage. Therefore, we employed a VE-cadherin promoter-driven CREERT2:THlox/THlox transgenic mouse line to ablate the tyrosine hydroxylase gene from endothelial cells in adult animals. After 26 weeks, but not 13 weeks, following the genetic blockade of tyrosine hydroxylase expression in VE-cadherin+ cells, we observed a significant reduction in tyrosine hydroxylase+ neurons in the substantia nigra. The results from this genetic lineage tracing study suggest that dopaminergic neurons are replenished in adult mice by a VE-cadherin+ progenitor cell population potentially arising from an endothelial lineage.

  18. Vomeronasal inputs to the rodent ventral striatum.

    Science.gov (United States)

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

    2008-03-18

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

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

    Science.gov (United States)

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

    2008-01-09

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

  20. The combination of ethanol with mephedrone increases the signs of neurotoxicity and impairs neurogenesis and learning in adolescent CD-1 mice

    Energy Technology Data Exchange (ETDEWEB)

    Ciudad-Roberts, Andrés; Duart-Castells, Leticia; Camarasa, Jorge; Pubill, David, E-mail: d.pubill@ub.edu; Escubedo, Elena

    2016-02-15

    A new family of psychostimulants, under the name of cathinones, has broken into the market in the last decade. In light of the fact that around 95% of cathinone consumers have been reported to combine them with alcoholic drinks, we sought to study the consequences of the concomitant administration of ethanol on mephedrone -induced neurotoxicity. Adolescent male Swiss-CD1 mice were administered four times in one day, every 2 h, with saline, mephedrone (25 mg/kg), ethanol (2; 1.5; 1.5; 1 g/kg) and their combination at a room temperature of 26 ± 2 °C. The combination with ethanol impaired mephedrone-induced decreases in dopamine transporter and tyrosine hydroxylase in the frontal cortex; and in serotonin transporter and tryptophan hydroxylase in the hippocampus by approximately 2-fold, 7 days post-treatment. Furthermore, these decreases correlated with a 2-fold increase in lipid peroxidation, measured as concentration of malondialdehyde (MDA), 24 h post-treatment, and were accompanied by changes in oxidative stress-related enzymes. Ethanol also notably potentiated mephedrone-induced negative effects on learning and memory, as well as hippocampal neurogenesis, measured through the Morris water maze (MWM) and 5-bromo-2′-deoxyuridine staining, respectively. These results are of special significance, since alcohol is widely co-abused with amphetamine derivatives such as mephedrone, especially during adolescence, a crucial stage in brain maturation. Given that the hippocampus is greatly involved in learning and memory processes, normal brain development in young adults could be affected with permanent behavioral consequences after this type of drug co-abuse. - Highlights: • Mice were administered a binge regimen of mephedrone plus/minus ethanol. • Ethanol exacerbated mephedrone-induced changes in 5-HT and DA function markers. • Neurochemical alterations were accompanied by an increase in oxidative stress. • Ethanol potentiated mephedrone-induced learning

  1. The combination of ethanol with mephedrone increases the signs of neurotoxicity and impairs neurogenesis and learning in adolescent CD-1 mice

    International Nuclear Information System (INIS)

    Ciudad-Roberts, Andrés; Duart-Castells, Leticia; Camarasa, Jorge; Pubill, David; Escubedo, Elena

    2016-01-01

    A new family of psychostimulants, under the name of cathinones, has broken into the market in the last decade. In light of the fact that around 95% of cathinone consumers have been reported to combine them with alcoholic drinks, we sought to study the consequences of the concomitant administration of ethanol on mephedrone -induced neurotoxicity. Adolescent male Swiss-CD1 mice were administered four times in one day, every 2 h, with saline, mephedrone (25 mg/kg), ethanol (2; 1.5; 1.5; 1 g/kg) and their combination at a room temperature of 26 ± 2 °C. The combination with ethanol impaired mephedrone-induced decreases in dopamine transporter and tyrosine hydroxylase in the frontal cortex; and in serotonin transporter and tryptophan hydroxylase in the hippocampus by approximately 2-fold, 7 days post-treatment. Furthermore, these decreases correlated with a 2-fold increase in lipid peroxidation, measured as concentration of malondialdehyde (MDA), 24 h post-treatment, and were accompanied by changes in oxidative stress-related enzymes. Ethanol also notably potentiated mephedrone-induced negative effects on learning and memory, as well as hippocampal neurogenesis, measured through the Morris water maze (MWM) and 5-bromo-2′-deoxyuridine staining, respectively. These results are of special significance, since alcohol is widely co-abused with amphetamine derivatives such as mephedrone, especially during adolescence, a crucial stage in brain maturation. Given that the hippocampus is greatly involved in learning and memory processes, normal brain development in young adults could be affected with permanent behavioral consequences after this type of drug co-abuse. - Highlights: • Mice were administered a binge regimen of mephedrone plus/minus ethanol. • Ethanol exacerbated mephedrone-induced changes in 5-HT and DA function markers. • Neurochemical alterations were accompanied by an increase in oxidative stress. • Ethanol potentiated mephedrone-induced learning

  2. Protein-Tyrosine Phosphorylation in Bacillus subtilis

    DEFF Research Database (Denmark)

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

    2005-01-01

    phosphorylation, indicating that this post-translational modifi cation could regulate physiological processes ranging from stress response and exopolysaccharide synthesis to DNA metabolism. Some interesting work in this fi eld was done in Bacillus subtilis , and we here present the current state of knowledge...... 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. Phosphorylation-mediated regulation of the Staphylococcus aureus secreted tyrosine phosphatase PtpA.

    Science.gov (United States)

    Brelle, Solène; Baronian, Grégory; Huc-Brandt, Sylvaine; Zaki, Laila Gannoun; Cohen-Gonsaud, Martin; Bischoff, Markus; Molle, Virginie

    2016-01-15

    Due to the emergence of methicillin-resistant strains, Staphylococcus aureus has become as major public-health threat. Studies aimed at deciphering the molecular mechanism of virulence are thus required to identify new targets and develop efficient therapeutic agents. Protein phosphorylations are known to play key regulatory functions and their roles in pathogenesis are under intense scrutiny. Here we analyzed the protein tyrosine phosphatase PtpA of S. aureus, a member of the family of low molecular weight protein tyrosine phosphatases that are often secreted by pathogenic bacteria. We report for the first time that PtpA is phosphorylated in vitro by the S. aureus tyrosine kinase CapA1B2. A mass spectrometry approach allowed determining that Tyr122 and Tyr123 were the only two residues phosphorylated by this kinase. This result was confirmed by analysis of a double PtpA_Y122A/Y123A mutant that showed no phosphorylation by CapA1B2. Interestingly, PtpA phosphatase activity was abrogated in this mutant, suggesting a key regulatory function for these two tyrosine residues. This was further reinforced by the observation that CapA1B2-mediated phosphorylation significantly increased PtpA phosphatase activity. Moreover, we provide evidence that PtpA is secreted during growth of S. aureus. Together our results suggest that PtpA is an exported S. aureus signaling molecule controlled by tyrosine phosphorylation which may interfere with host cell signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. 25-Hydroxyvitamin D depletion does not exacerbate MPTP-induced dopamine neuron damage in mice.

    Directory of Open Access Journals (Sweden)

    E Danielle Dean

    Full Text Available Recent clinical evidence supports a link between 25-hydroxyvitamin D insufficiency (serum 25-hydroxyvitamin D [25(OHD] levels <30 ng/mL and Parkinson's disease. To investigate the effect of 25(OHD depletion on neuronal susceptibility to toxic insult, we induced a state of 25(OHD deficiency in mice and then challenged them with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP. We found there was no significant difference between control and 25(OHD-deficient animals in striatal dopamine levels or dopamine transporter and tyrosine hydroxylase expression after lesioning with MPTP. Additionally, we found no difference in tyrosine hydroxylase expression in the substantia nigra pars compacta. Our data suggest that reducing 25(OHD serum levels in mice has no effect on the vulnerability of nigral dopaminergic neurons in vivo in this model system of parkinsonism.

  5. Sex-dependent differences in phenobarbitane-induced oestradiol-2-hydroxylase activity in rat liver

    Energy Technology Data Exchange (ETDEWEB)

    Theron, C.N.; Neethling, A.C.; Taljaard, J.J.F. (Stellenbosch Univ. (South Africa). Dept. of Chemical Pathology)

    1981-08-15

    Oestradiol-2-hydroxylase (E/sub 2/-OH) activity was measured in liver and brain microsomes of 6-8-week-old Wistar rats. Phenobarbitone (75 mg/kg daily for 3 days) significantly increased enzyme activity in the liver of males and females, but there were striking differences between the two sexes. In males the enzyme activity was increased by 37% over control values and in females by 200%. The total microsomol cytochrome P-450 content was increased by 75% in males and by 82% in females. The apparent Michaelis constant (K(m)) of E/sub 2/-OH for 17..beta..-oestradiol in untreated males (9,8 ..mu..M) and females (9,2 ..mu..M) did not differ significantly. Phenobarbitone treatment, however, tended to reduce the apparent K(m) in males (8,2 ..mu..M) and to increase it in females (18,7 ..mu..M). E/sub 2/-OH activity was also detected in brain tissue of both sexes, but it was 50-200-fold lower than in the liver and was not increased by phenobarbitone.

  6. Sex-dependent differences in phenobarbitane-induced oestradiol-2-hydroxylase activity in rat liver

    International Nuclear Information System (INIS)

    Theron, C.N.; Neethling, A.C.; Taljaard, J.J.F.

    1981-01-01

    Oestradiol-2-hydroxylase (E 2 -OH) activity was measured in liver and brain microsomes of 6-8-week-old Wistar rats. Phenobarbitone (75 mg/kg daily for 3 days) significantly increased enzyme activity in the liver of males and females, but there were striking differences between the two sexes. In males the enzyme activity was increased by 37% over control values and in females by 200%. The total microsomol cytochrome P-450 content was increased by 75% in males and by 82% in females. The apparent Michaelis constant (K(m)) of E 2 -OH for 17β-oestradiol in untreated males (9,8 μM) and females (9,2 μM) did not differ significantly. Phenobarbitone treatment, however, tended to reduce the apparent K(m) in males (8,2 μM) and to increase it in females (18,7 μM). E 2 -OH activity was also detected in brain tissue of both sexes, but it was 50-200-fold lower than in the liver and was not increased by phenobarbitone

  7. Effect of acute maternal starvation on tyrosine metabolism and protein synthesis in fetal sheep

    International Nuclear Information System (INIS)

    Krishnamurti, C.R.; Schaefer, A.L.

    1984-01-01

    To determine the effects of acute maternal starvation on intrauterine growth, tyrosine concentration and specific activity values in plasma, intracellular free and protein bound pools were determined in catheterized ovine fetuses following an 8 h continuous infusion of L-[2,3,5,6 3 H] or L-[U- 14 C] tyrosine into the ewe and fetus respectively at 115-125 days of gestation. From the kinetic data the rates of whole body and tissue fractional protein synthesis were calculated. Although placental protein synthesis was not significantly changed as a result of acute maternal starvation, fetal whole body protein synthesis was reduced from 63 g/d/kg in the fed to 25 g/d/kg in the starved condition. There was also a 10 fold reduction in the net placental transfer of tyrosine to the fetus in the starved ewes. In addition, a three fold increase was observed in the quantity of tyrosine used for oxidation by the fetuses of starved ewes, changing from 5.2% of tyrosine net utilization in the fed to 13.7% in the starved condition. Significant reductions in tissue fractional protein synthesis rates were also seen in the liver, brain, lung kidney and GIT tissues from 78, 37, 65, 45 and 71%/d respectively in the fed to 12, 10, 23, 22 and 35%/d in the fetuses of starved ewes. The data indicate that during acute maternal starvation the sheep fetus utilizes more tyrosine for oxidation and less for anabolic purposes which is reflected in a decrease both in whole body and tissue fractional rates of protein synthesis

  8. Ror receptor tyrosine kinases: orphans no more

    OpenAIRE

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

    2008-01-01

    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 act...

  9. Increased activity of the Vesicular Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor TI-VAMP/VAMP7 by Tyrosine Phosphorylation in the Longin Domain*

    Science.gov (United States)

    Burgo, Andrea; Casano, Alessandra M.; Kuster, Aurelia; Arold, Stefan T.; Wang, Guan; Nola, Sébastien; Verraes, Agathe; Dingli, Florent; Loew, Damarys; Galli, Thierry

    2013-01-01

    Vesicular (v)- and target (t)-SNAREs play essential roles in intracellular membrane fusion through the formation of cytoplasmic α-helical bundles. Several v-SNAREs have a Longin N-terminal extension that, by promoting a closed conformation, plays an autoinhibitory function and decreases SNARE complex formation and membrane fusion efficiency. The molecular mechanism leading to Longin v-SNARE activation is largely unknown. Here we find that exocytosis mediated by the Longin v-SNARE TI-VAMP/VAMP7 is activated by tonic treatment with insulin and insulin-like growth factor-1 but not by depolarization and intracellular calcium rise. In search of a potential downstream mechanism, we found that TI-VAMP is phosphorylated in vitro by c-Src kinase on tyrosine 45 of the Longin domain. Accordingly, a mutation of tyrosine 45 into glutamate, but not phenylalanine, activates both t-SNARE binding and exocytosis. Activation of TI-VAMP-mediated exocytosis thus relies on tyrosine phosphorylation. PMID:23471971

  10. 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.

  11. Tyrosine-like condensed derivatives as tyrosinase inhibitors.

    Science.gov (United States)

    Matos, Maria João; Santana, Lourdes; Uriarte, Eugenio; Serra, Silvia; Corda, Marcella; Fadda, Maria Benedetta; Era, Benedetta; Fais, Antonella

    2012-05-01

    We report the pharmacological evaluation of a new series of 3-aminocoumarins differently substituted with hydroxyl groups, which have been synthesized because they include in their structures the tyrosine fragment (tyrosine-like compounds), with the aim of discovering structural features necessary for tyrosinase inhibitory activity. The synthesized compounds 4 and 7-9 were evaluated in vitro as mushroom tyrosinase inhibitors. Two of the described compounds showed lower IC50 (concentration giving 50% inhibition of tyrosinase activity) than umbelliferone, used as a reference compound. Compound 7 (IC50=53µm) was the best tyrosinase inhibitor of this small series, having an IC50 value 10-fold lower than umbelliferone. Compound 7 (3-amino-7-hydroxycoumarin) had amino and hydroxyl groups precisely mimicking the same positions that both groups occupy on the tyrosine molecule. © 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society.

  12. 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...

  13. Determination of o-tyrosine in shrimps, fish, mussels and egg-white

    International Nuclear Information System (INIS)

    Meier, W.; Hediger, H.; Artho, A.; Meier, E.J.M.

    1993-01-01

    With this new HPLC-system the o-tyrosine in irradiated shrimps, fish, mussels and egg-white is very well separated from other peaks in the chromatogram. It is not any more necessary to freeze dry the samples. Samples with an amount of o-tyrosine greater than 0.1 mg/kg are suspect. To confirm such results, the o-tyrosine fraction can be collected and the o-tyrosine can be determined either by GC/MS after derivatisation with chloro-formicacid-methylester or by a second HPLC-step using a cation exchange column. (orig./vhe)

  14. Browse Title Index

    African Journals Online (AJOL)

    Items 101 - 150 of 273 ... Vol 32 (2006):, Lead pollution in urban roadside environments of Dar es ... Vol 30 (2004): Special Issue on Lake Victoria, Levels of nitrate and ... of Tyrosine Hydroxylase Immunoreactive Cells in the Mouse Islets of ...

  15. Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung.

    Science.gov (United States)

    Martini, Sabrina V; Silva, Adriana L; Ferreira, Debora; Rabelo, Rafael; Ornellas, Felipe M; Gomes, Karina; Rocco, Patricia R M; Petrs-Silva, Hilda; Morales, Marcelo M

    2016-01-01

    Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response. Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy. © 2016 The Author(s) Published by S. Karger AG, Basel.

  16. 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...

  17. Receptor tyrosine kinase signaling: a view from quantitative proteomics

    DEFF Research Database (Denmark)

    Dengjel, Joern; Kratchmarova, Irina; Blagoev, Blagoy

    2009-01-01

    Growth factor receptor signaling via receptor tyrosine kinases (RTKs) is one of the basic cellular communication principals found in all metazoans. Extracellular signals are transferred via membrane spanning receptors into the cytoplasm, reversible tyrosine phosphorylation being the hallmark of all...

  18. Testing whether Metazoan Tyrosine Loss Was Driven by Selection against Promiscuous Phosphorylation

    Science.gov (United States)

    Pandya, Siddharth; Struck, Travis J.; Mannakee, Brian K.; Paniscus, Mary; Gutenkunst, Ryan N.

    2015-01-01

    Protein tyrosine phosphorylation is a key regulatory modification in metazoans, and the corresponding kinase enzymes have diversified dramatically. This diversification is correlated with a genome-wide reduction in protein tyrosine content, and it was recently suggested that this reduction was driven by selection to avoid promiscuous phosphorylation that might be deleterious. We tested three predictions of this intriguing hypothesis. 1) Selection should be stronger on residues that are more likely to be phosphorylated due to local solvent accessibility or structural disorder. 2) Selection should be stronger on proteins that are more likely to be promiscuously phosphorylated because they are abundant. We tested these predictions by comparing distributions of tyrosine within and among human and yeast orthologous proteins. 3) Selection should be stronger against mutations that create tyrosine versus remove tyrosine. We tested this prediction using human population genomic variation data. We found that all three predicted effects are modest for tyrosine when compared with the other amino acids, suggesting that selection against deleterious phosphorylation was not dominant in driving metazoan tyrosine loss. PMID:25312910

  19. Characterization of the beta-carotene hydroxylase gene DSM2 conferring drought and oxidative stress resistance by increasing xanthophylls and abscisic acid synthesis in rice.

    Science.gov (United States)

    Du, Hao; Wang, Nili; Cui, Fei; Li, Xianghua; Xiao, Jinghua; Xiong, Lizhong

    2010-11-01

    Drought is a major limiting factor for crop production. To identify critical genes for drought resistance in rice (Oryza sativa), we screened T-DNA mutants and identified a drought-hypersensitive mutant, dsm2. The mutant phenotype was caused by a T-DNA insertion in a gene encoding a putative β-carotene hydroxylase (BCH). BCH is predicted for the biosynthesis of zeaxanthin, a carotenoid precursor of abscisic acid (ABA). The amounts of zeaxanthin and ABA were significantly reduced in two allelic dsm2 mutants after drought stress compared with the wild type. Under drought stress conditions, the mutant leaves lost water faster than the wild type and the photosynthesis rate, biomass, and grain yield were significantly reduced, whereas malondialdehyde level and stomata aperture were increased in the mutant. The mutant is also hypersensitive to oxidative stresses. The mutant had significantly lower maximal efficiency of photosystem II photochemistry and nonphotochemical quenching capacity than the wild type, indicating photoinhibition in photosystem II and decreased capacity for eliminating excess energy by thermal dissipation. Overexpression of DSM2 in rice resulted in significantly increased resistance to drought and oxidative stresses and increases of the xanthophylls and nonphotochemical quenching. Some stress-related ABA-responsive genes were up-regulated in the overexpression line. DSM2 is a chloroplast protein, and the response of DSM2 to environmental stimuli is distinctive from the other two BCH members in rice. We conclude that the DSM2 gene significantly contributes to control of the xanthophyll cycle and ABA synthesis, both of which play critical roles in the establishment of drought resistance in rice.

  20. 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.

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

    Science.gov (United States)

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

    2018-04-03

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

  2. 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.

  3. Elevated Serum Levels of Cysteine and Tyrosine: Early Biomarkers in Asymptomatic Adults at Increased Risk of Developing Metabolic Syndrome

    Directory of Open Access Journals (Sweden)

    Nina Mohorko

    2015-01-01

    Full Text Available As there is effective intervention for delaying or preventing metabolic diseases, which are often present for years before becoming clinically apparent, novel biomarkers that would mark metabolic complications before the onset of metabolic disease should be identified. We investigated the role of fasting serum amino acids and their associations with inflammatory markers, adipokines, and metabolic syndrome (MetS components in subjects prior to the onset of insulin resistance (IR. Anthropometric measurements, food records, adipokines, biochemical markers, and serum levels of amino acids were determined in 96 asymptomatic subjects aged 25–49 years divided into three groups according to the number of MetS components present. Cysteine and tyrosine were significantly higher already in group with one component of MetS present compared to subjects without MetS components. Serum amino acid levels correlated with markers of inflammation and adipokines. Alanine and glycine explained 10% of insulin resistance variability. The role of tyrosine and cysteine, that were higher already with 1 component of MetS present, should be further investigated as they might point to future insulin disturbances.

  4. 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

  5. Enhancement of L-phenylalanine production by engineered Escherichia coli using phased exponential L-tyrosine feeding combined with nitrogen source optimization.

    Science.gov (United States)

    Yuan, Peipei; Cao, Weijia; Wang, Zhen; Chen, Kequan; Li, Yan; Ouyang, Pingkai

    2015-07-01

    Nitrogen source optimization combined with phased exponential L-tyrosine feeding was employed to enhance L-phenylalanine production by a tyrosine-auxotroph strain, Escherichia coli YP1617. The absence of (NH4)2SO4, the use of corn steep powder and yeast extract as composite organic nitrogen source were more suitable for cell growth and L-phenylalanine production. Moreover, the optimal initial L-tyrosine level was 0.3 g L(-1) and exponential L-tyrosine feeding slightly improved L-phenylalanine production. Nerveless, L-phenylalanine production was greatly enhanced by a strategy of phased exponential L-tyrosine feeding, where exponential feeding was started at the set specific growth rate of 0.08, 0.05, and 0.02 h(-1) after 12, 32, and 52 h, respectively. Compared with exponential L-tyrosine feeding at the set specific growth rate of 0.08 h(-1), the developed strategy obtained a 15.33% increase in L-phenylalanine production (L-phenylalanine of 56.20 g L(-1)) and a 45.28% decrease in L-tyrosine supplementation. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. Singlet oxygen generation during the oxidation of L-tyrosine and L-dopa with mushroom tyrosinase

    Energy Technology Data Exchange (ETDEWEB)

    Miyaji, Akimitsu [Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259-G1-14, Nagatsuta-cho, Midori-ku, Yokohama 226-8502 (Japan); Kohno, Masahiro [Department of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259-G1-25 Nagatsuta-cho, Midori-ku, Yokohama 226-8502 (Japan); Inoue, Yoshihiro [Showa Pharmaceutical University, 3-3165 Higashi-tamagawagakuen, Machida, Tokyo 194-8543 (Japan); Baba, Toshihide, E-mail: tbaba@chemenv.titech.ac.jp [Department of Environmental Chemistry and Engineering, Tokyo Institute of Technology, 4259-G1-14, Nagatsuta-cho, Midori-ku, Yokohama 226-8502 (Japan)

    2016-03-18

    The generation of singlet oxygen during the oxidation of tyrosine and L-dopa using mushroom tyrosinase in a phosphate buffer (pH 7.4), the model of melanin synthesis in melanocytes, was examined. The reaction was performed in the presence of 2,2,6,6-tetramethyl-4-piperidone (4-oxo-TEMP), an acceptor of singlet oxygen and the electron spin resonance (ESR) of the spin adduct, 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO), was measured. An increase in the ESR signal attributable to 4-oxo-TEMPO was observed during the oxidation of tyrosine and L-dopa with tyrosinase, indicating the generation of singlet oxygen. The results suggest that {sup 1}O{sub 2} generation via tyrosinase-catalyzed melanin synthesis occurs in melanocyte. - Highlights: • Generation of singlet oxygen was observed during tyrosinase-catalyzed tyrosine oxidation. • The singlet oxygen generated when tyrosine was converted into dopachrome. • The amount of singlet oxygen is not sufficient for cell toxicity. • It decreased when the hydroxyl radicals and/or superoxide anions were trapped.

  7. Characterization of a Dopaminergic Stimulatory Factor Derived from Monoclonal Cell Lines of Striatal Origin

    Science.gov (United States)

    2006-12-01

    dissolved in dimethylsulfoxide ( DMSO ). Dopamine level of MSO vehicle control = 69 ng/mg protein.he presence of a carboxylic acid group and the...phosphoethanolamine. The second, in contrast, could be extracted by organic solvents from a concentrate of the X61 lysate and was found after purification to...neurotransmitter stores by alpha-methy-p-tyrosine (AMT), an inhibitor of tyrosine hydroxylase. MN9D and N18TG2 cells were treated for 48 hrs with DMSO

  8. TATN-1 mutations reveal a novel role for tyrosine as a metabolic signal that influences developmental decisions and longevity in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Annabel A Ferguson

    Full Text Available Recent work has identified changes in the metabolism of the aromatic amino acid tyrosine as a risk factor for diabetes and a contributor to the development of liver cancer. While these findings could suggest a role for tyrosine as a direct regulator of the behavior of cells and tissues, evidence for this model is currently lacking. Through the use of RNAi and genetic mutants, we identify tatn-1, which is the worm ortholog of tyrosine aminotransferase and catalyzes the first step of the conserved tyrosine degradation pathway, as a novel regulator of the dauer decision and modulator of the daf-2 insulin/IGF-1-like (IGFR signaling pathway in Caenorhabditis elegans. Mutations affecting tatn-1 elevate tyrosine levels in the animal, and enhance the effects of mutations in genes that lie within the daf-2/insulin signaling pathway or are otherwise upstream of daf-16/FOXO on both dauer formation and worm longevity. These effects are mediated by elevated tyrosine levels as supplemental dietary tyrosine mimics the phenotypes produced by a tatn-1 mutation, and the effects still occur when the enzymes needed to convert tyrosine into catecholamine neurotransmitters are missing. The effects on dauer formation and lifespan require the aak-2/AMPK gene, and tatn-1 mutations increase phospho-AAK-2 levels. In contrast, the daf-16/FOXO transcription factor is only partially required for the effects on dauer formation and not required for increased longevity. We also find that the controlled metabolism of tyrosine by tatn-1 may function normally in dauer formation because the expression of the TATN-1 protein is regulated both by daf-2/IGFR signaling and also by the same dietary and environmental cues which influence dauer formation. Our findings point to a novel role for tyrosine as a developmental regulator and modulator of longevity, and support a model where elevated tyrosine levels play a causal role in the development of diabetes and cancer in people.

  9. TATN-1 Mutations Reveal a Novel Role for Tyrosine as a Metabolic Signal That Influences Developmental Decisions and Longevity in Caenorhabditis elegans

    Science.gov (United States)

    Ferguson, Annabel A.; Dumas, Kathleen J.; Ritov, Vladimir B.; Matern, Dietrich; Hu, Patrick J.; Fisher, Alfred L.

    2013-01-01

    Recent work has identified changes in the metabolism of the aromatic amino acid tyrosine as a risk factor for diabetes and a contributor to the development of liver cancer. While these findings could suggest a role for tyrosine as a direct regulator of the behavior of cells and tissues, evidence for this model is currently lacking. Through the use of RNAi and genetic mutants, we identify tatn-1, which is the worm ortholog of tyrosine aminotransferase and catalyzes the first step of the conserved tyrosine degradation pathway, as a novel regulator of the dauer decision and modulator of the daf-2 insulin/IGF-1-like (IGFR) signaling pathway in Caenorhabditis elegans. Mutations affecting tatn-1 elevate tyrosine levels in the animal, and enhance the effects of mutations in genes that lie within the daf-2/insulin signaling pathway or are otherwise upstream of daf-16/FOXO on both dauer formation and worm longevity. These effects are mediated by elevated tyrosine levels as supplemental dietary tyrosine mimics the phenotypes produced by a tatn-1 mutation, and the effects still occur when the enzymes needed to convert tyrosine into catecholamine neurotransmitters are missing. The effects on dauer formation and lifespan require the aak-2/AMPK gene, and tatn-1 mutations increase phospho-AAK-2 levels. In contrast, the daf-16/FOXO transcription factor is only partially required for the effects on dauer formation and not required for increased longevity. We also find that the controlled metabolism of tyrosine by tatn-1 may function normally in dauer formation because the expression of the TATN-1 protein is regulated both by daf-2/IGFR signaling and also by the same dietary and environmental cues which influence dauer formation. Our findings point to a novel role for tyrosine as a developmental regulator and modulator of longevity, and support a model where elevated tyrosine levels play a causal role in the development of diabetes and cancer in people. PMID:24385923

  10. Membrane depolarization-induced RhoA/Rho-associated kinase activation and sustained contraction of rat caudal arterial smooth muscle involves genistein-sensitive tyrosine phosphorylation

    Science.gov (United States)

    Mita, Mitsuo; Tanaka, Hitoshi; Yanagihara, Hayato; Nakagawa, Jun-ichi; Hishinuma, Shigeru; Sutherland, Cindy; Walsh, Michael P.; Shoji, Masaru

    2013-01-01

    Rho-associated kinase (ROK) activation plays an important role in K+-induced contraction of rat caudal arterial smooth muscle (Mita et al., Biochem J. 2002; 364: 431–40). The present study investigated a potential role for tyrosine kinase activity in K+-induced RhoA activation and contraction. The non-selective tyrosine kinase inhibitor genistein, but not the src family tyrosine kinase inhibitor PP2, inhibited K+-induced sustained contraction (IC50 = 11.3 ± 2.4 µM). Genistein (10 µM) inhibited the K+-induced increase in myosin light chain (LC20) phosphorylation without affecting the Ca2+ transient. The tyrosine phosphatase inhibitor vanadate induced contraction that was reversed by genistein (IC50 = 6.5 ± 2.3 µM) and the ROK inhibitor Y-27632 (IC50 = 0.27 ± 0.04 µM). Vanadate also increased LC20 phosphorylation in a genistein- and Y-27632-dependent manner. K+ stimulation induced translocation of RhoA to the membrane, which was inhibited by genistein. Phosphorylation of MYPT1 (myosin-targeting subunit of myosin light chain phosphatase) was significantly increased at Thr855 and Thr697 by K+ stimulation in a genistein- and Y-27632-sensitive manner. Finally, K+ stimulation induced genistein-sensitive tyrosine phosphorylation of proteins of ∼55, 70 and 113 kDa. We conclude that a genistein-sensitive tyrosine kinase, activated by the membrane depolarization-induced increase in [Ca2+]i, is involved in the RhoA/ROK activation and sustained contraction induced by K+. Ca2+ sensitization, myosin light chain phosphatase, RhoA, Rho-associated kinase, tyrosine kinase PMID:24133693

  11. Manganese superoxide dismutase (MnSOD catalyzes NO-dependent tyrosine residue nitration

    Directory of Open Access Journals (Sweden)

    SRDJAN STOJANOVIC

    2005-04-01

    Full Text Available The peroxynitrite-induced nitration of manganese superoxide dismutase (MnSOD tyrosine residue, which causes enzyme inactivation, is well established. This led to suggestions that MnSOD nitration and inactivation in vivo, detected in various diseases associated with oxidative stress and overproduction of nitric monoxide (NO, conditions which favor peroxynitrite formation, is also caused by peroxynitrite. However, our previous in vitro study demonstrated that exposure of MnSOD to NO led to NO conversion into nitrosonium (NO+ and nitroxyl (NO– species, which caused enzyme modifications and inactivation. Here it is reported that MnSOD is tyrosine nitrated upon exposure to NO, as well as that MnSOD nitration contributes to inactivation of the enzyme. Collectively, these observations provide a compelling argument supporting the generation of nitrating species in MnSOD exposed to NO and shed a new light on MnSOD tyrosine nitration and inactivation in vivo. This may represent a novel mechanism by which MnSOD protects cell from deleterious effects associated with overproduction of NO. However, extensive MnSOD modification and inactivation associated with prolonged exposure to NO will amplify the toxic effects caused by increased cell superoxide and NO levels.

  12. 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.

  13. Ret function in muscle stem cells points to tyrosine kinase inhibitor therapy for facioscapulohumeral muscular dystrophy.

    Science.gov (United States)

    Moyle, Louise A; Blanc, Eric; Jaka, Oihane; Prueller, Johanna; Banerji, Christopher Rs; Tedesco, Francesco Saverio; Harridge, Stephen Dr; Knight, Robert D; Zammit, Peter S

    2016-11-14

    Facioscapulohumeral muscular dystrophy (FSHD) involves sporadic expression of DUX4, which inhibits myogenesis and is pro-apoptotic. To identify target genes, we over-expressed DUX4 in myoblasts and found that the receptor tyrosine kinase Ret was significantly up-regulated, suggesting a role in FSHD. RET is dynamically expressed during myogenic progression in mouse and human myoblasts. Constitutive expression of either RET9 or RET51 increased myoblast proliferation, whereas siRNA-mediated knockdown of Ret induced myogenic differentiation. Suppressing RET activity using Sunitinib, a clinically-approved tyrosine kinase inhibitor, rescued differentiation in both DUX4-expressing murine myoblasts and in FSHD patient-derived myoblasts. Importantly, Sunitinib also increased engraftment and differentiation of FSHD myoblasts in regenerating mouse muscle. Thus, DUX4-mediated activation of Ret prevents myogenic differentiation and could contribute to FSHD pathology by preventing satellite cell-mediated repair. Rescue of DUX4-induced pathology by Sunitinib highlights the therapeutic potential of tyrosine kinase inhibitors for treatment of FSHD.

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

    Science.gov (United States)

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

    2016-07-01

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

  15. The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration

    DEFF Research Database (Denmark)

    Voena, Claudia; Conte, Chiara; Ambrogio, Chiara

    2007-01-01

    Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades......, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine...... phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542...

  16. Synthesis of [N-C[sup 3]H[sub 3

    Energy Technology Data Exchange (ETDEWEB)

    Wyrick, S D; Myers, A M; Booth, R G; Kula, N S; Baldessarini, R J; Mailman, R B [North Carolina Univ., Chapel Hill, NC (United States). School of Pharmacy and Brain and Development Research Center Harvard Medical School, Boston, MA (United States) Massachusetts General Hospital, Belmont, MA (United States). Mailman Research Center

    1994-02-01

    Subsequent to the discovery that the (+)-benzomorphan sigma receptor ligands, (+)-pentazocine and (+)-N-allylnormetazocine, stimulated tyrosine hydroxylase activity and dopamine synthesis in rat striatum in vitro, we reported a similar effect on a structurally similar series of 1-phenyl-3-aminotetrahydronaphthalenes (phenylaminotetralins, PAT's). We previously reported the synthesis of tritium labeled Cl,OH-PAT to be used in radioreceptor and autoradiography studies and found that it labeled a sigma-like site in guinea pig brain with an apparent Kd of [approx] 50 pM and with a pharmacological profile unique from other known CNS receptors. Here we report the synthesis of high specific activity tritium labeled trans-(1R,3S)-(-)-H[sub 2]PAT as this enantiomer was found to be more active in the tyrosine hydroxylase assay and possessed approximately 45 fold greater affinity for the novel neuromodulatory sigma-like receptor. (author).

  17. Tracking neuronal marker expression inside living differentiating cells using molecular beacons

    DEFF Research Database (Denmark)

    Ilieva, Mirolyuba; Della Vedova, Paolo; Hansen, Ole

    2013-01-01

    and tyrosine hydroxylase mRNAs were expressed 2 and 3 days post induction of differentiation, respectively. Oct 4 was not detected with MB in these cells and signal was not increased over time suggesting that MB are generally stable inside the cells. The gene expression changes measured using MBs were...... confirmed using qRT-PCR. These results suggest that MBs are simple to use sensors inside living cell, and particularly useful for studying dynamic gene expression in heterogeneous cell populations....

  18. Study of interaction between tryptophan, tyrosine, and phenylalanine separately with silver nanoparticles by fluorescence quenching method

    International Nuclear Information System (INIS)

    Roy, S.; Das, T.K.

    2015-01-01

    Using the spectroscopic method, the individual interaction of the three biochemically important amino acids, which are constituents of protein, namely, tryptophan, tyrosine, and phenylalanine with biologically synthesized silver nanoparticles has been investigated. The obtained UV-Vis spectra show the formation of ground-state complexes between tryptophan, tyrosine, and phenylalanine with silver nanoparticles. Silver nanoparticles possess the ability to quench the intrinsic fluorescence of the aforesaid amino acids by a dynamic quenching process. The binding constant, number of binding sites, and corresponding thermodynamic parameters (ΔH, ΔS, and ΔG) based on the interaction system were calculated for 293, 303, and 313 K. In the case of tryptophan and phenylalanine, with increase in temperature, the binding constant K was found to decrease; conversely, it was found to increase with increase in temperature in the case of tyrosine. The thermodynamic results revealed that the binding process was spontaneous; hydrogen bonding and van der Waals interaction were the predominant forces responsible for the complex stabilization in the case of tryptophan and phenylalanine, respectively, whereas in the case of tyrosine, hydrophobic interaction was the sole force conferring stability. Moreover, the Förster non-radiation energy transfer theory has been applied to calculate the average binding distance among the above amino acids and silver nanoparticles. The results show a binding distance of <7 nm, which ensures that energy transfer does occur between the said amino acids and silver nanoparticles. (authors)

  19. Determination of o-tyrosine as a marker for the detection of irradiated shrimps

    International Nuclear Information System (INIS)

    Hunková, J.; Simat, T.J.; Steinhart, H.

    2000-01-01

    o-tyrosine is proposed as a marker for the identification of irradiated protein-rich food. An HPLC method for qualitative and quantitative determination of non-protein bound o-tyrosine in shrimps (Crangon crangon) has been developed. For this purpose the o-tyrosine was extracted from non-irradiated as well as irradiated samples with perchloric acid, then separated isocratically (ammoniumformiat buffer, pH 4) on an RP-C18 column and detected by FLD (275/305 nm). The quantification of o-tyrosine was based on the use of alfa-methyl-p-tyrosine as internal standard. In non-irradiated shrimps a background level of 28.9 microg/kg was found. The content of o-tyrosine in 1 kGy irradiated shrimps was found to be 119.9 mikrog/kg, which was well 4-fold over the background level. The dependency between radiation dose and the amount of o-tyrosine was observed in the range of 0-5 kGy

  20. Protein tyrosine phosphatases: regulatory mechanisms.

    NARCIS (Netherlands)

    den Hertog, J.; Ostman, A.; Bohmer, F.D.

    2008-01-01

    Protein-tyrosine phosphatases are tightly controlled by various mechanisms, ranging from differential expression in specific cell types to restricted subcellular localization, limited proteolysis, post-translational modifications affecting intrinsic catalytic activity, ligand binding and

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

    Science.gov (United States)

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

    2015-01-05

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

  2. Protein tyrosine phosphatase receptor type R deficient mice exhibit increased exploration in a new environment and impaired novel object recognition memory

    NARCIS (Netherlands)

    Erkens, M.; Bakker, B.; Duijn, L.M. van; Hendriks, W.J.A.J.; Zee, C.E.E.M. van der

    2014-01-01

    Mouse gene Ptprr encodes multiple protein tyrosine phosphatase receptor type R (PTPRR) isoforms that negatively regulate mitogen-activated protein kinase (MAPK) signaling pathways. In the mouse brain, PTPRR proteins are expressed in cerebellum, olfactory bulb, hippocampus, amygdala and perirhinal

  3. Modular Engineering of l-Tyrosine Production in Escherichia coli

    Science.gov (United States)

    Juminaga, Darmawi; Baidoo, Edward E. K.; Redding-Johanson, Alyssa M.; Batth, Tanveer S.; Burd, Helcio; Mukhopadhyay, Aindrila; Petzold, Christopher J.

    2012-01-01

    Efficient biosynthesis of l-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for l-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to l-tyrosine on two plasmids. Rational engineering to improve l-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to l-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter l-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways. PMID:22020510

  4. Neurotransmitters and putative neuromodulators in the gut of Anguilla anguilla (L.. Localizations in the enteric nervous and endocrine systems

    Directory of Open Access Journals (Sweden)

    A Veggetti

    2009-12-01

    Full Text Available The gut of silver eels (Anguilla anguilla L. was investigated in order to describe both the cholinergic and adrenergic intramural innervations, and the localization of possible accessory neuromediators. Histochemical reactions for the demonstration of nicotinamide adenine dinucleotide phosphate, reduced form-(NADPH-diaphorase and acetylcholinesterese (AChEase were performed, as well as the immunohistochemical testing of tyrosine hydroxylase, met-enkephalin, substance P, calcitonin gene-related peptide (CGRP, bombesin, vasoactive intestinal peptide (VIP, neuropeptide Y (NPY, somatostatin, cholecystokinin-octapeptide (CCK-8, serotonin, cholineacetyltransferase. The results evidenced a different pattern in comparison with other vertebrates, namely mammals, and with other fish. Both NADPH-diaphorase and AChEase activities were histochemically detected all along the gut in the myenteric plexus, the inner musculature and the propria-submucosa. Tyrosine hydroxylase immunoreactivity was observed in the intestinal tract only, both in the myenteric plexus and in the inner musculature. Several neuropeptides (metenkephalin, CGRP, bombesin, substance P, VIP, NPY, somatostatin were, in addition, detected in the intramural innervation; some of them also in epithelial cells of the diffuse endocrine system (met-enkephalin, substance P, NPY, somatostatin. Serotonin was only present in endocrine cells. Tyrosine hydroxylase immunoreactivity was present in localizations to those of similar NADPHdiaphorase- reactivity, and in the same nerve bundles in which substance P- and CGRP-likeimmunoreactivities were detectable in the intestinal tract. In addition, NADPH-diaphorase-reactive neurons showed an anatomical relationship with AChEase-reactive nerve terminals, and a similar relationship existed between the latter and substance P-like immunoreactivity.

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

    Science.gov (United States)

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

    2018-02-01

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

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

    Science.gov (United States)

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

    2009-09-15

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

  7. Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase.

    Science.gov (United States)

    Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Liu, Ming C; Shetty, Sreerama

    2010-02-01

    The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1-100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.

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

    Science.gov (United States)

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

    2012-01-01

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

  9. The presence of tyrosine glucoside in the haemolymph of lepidopteran insects

    International Nuclear Information System (INIS)

    Ishizaki, Yumi; Umebachi, Yoshishige

    1980-01-01

    A ninhydrin-positive substance from the haemolymph of Papilio xuthus was purified and identified as β-glucosyl-O-tyrosine by (1) color reactions, (2) incorporation of 14 C-tyrosine, (3) identification and estimation of hydrolysis products, (4) α- and β-glucosidase tests, and (5) UV-spectrum. The concentration of the tyrosine glucoside in haemolymph reaches a maximum at the prepupal stage, then decreases, and is on a low level during the middle stage of pupa. At the late pupal stage, the level again rises and is kept high before emergence. After emergence, it rapidly decreases. The same tyrosine glucoside has proved to be also present in the haemolymph of twelve other species of Lepidoptera. (author)

  10. [Development and Application of Catalytic Tyrosine Modification].

    Science.gov (United States)

    Sato, Shinichi; Tsushima, Michihiko; Nakamura, Kosuke; Nakamura, Hiroyuki

    2018-01-01

     The chemical labeling of proteins with synthetic probes is a key technique used in chemical biology, protein-based therapy, and material science. Much of the chemical labeling of native proteins, however, depends on the labeling of lysine and cysteine residues. While those methods have significantly contributed to native protein labeling, alternative methods that can modify different amino acid residues are still required. Herein we report the development of a novel methodology of tyrosine labeling, inspired by the luminol chemiluminescence reaction. Tyrosine residues are often exposed on a protein's surface and are thus expected to be good targets for protein functionalization. In our studies so far, we have found that 1) hemin oxidatively activates luminol derivatives as a catalyst, 2) N-methyl luminol derivative specifically forms a covalent bond with a tyrosine residue among the 20 kinds of natural amino acid residues, and 3) the efficiency of tyrosine labeling with N-methyl luminol derivative is markedly improved by using horseradish peroxidase (HRP) as a catalyst. We were able to use molecular oxygen as an oxidant under HRP/NADH conditions. By using these methods, the functionalization of purified proteins was carried out. Because N-methyl luminol derivative is an excellent protein labeling reagent that responds to the activation of peroxidase, this new method is expected to open doors to such biological applications as the signal amplification of HRP-conjugated antibodies and the detection of protein association in combination with peroxidase-tag technology.

  11. Suppression of adhesion-induced protein tyrosine phosphorylation decreases invasive and metastatic potentials of B16-BL6 melanoma cells by protein tyrosine kinase inhibitor genistein.

    Science.gov (United States)

    Yan, C; Han, R

    1997-01-01

    Protein tyrosine kinase (PTK) appears to be involved in the activation of signaling during cell attachment to and spreading on extracellular matrix (ECM) in the metastatic cascade. To verify the assumption that PTK inhibitors might impair ECM signaling and prevent cancer metastasis, the highly metastatic B16-BL6 mouse melanoma cells were exposed to the PTK inhibitor genistein for 3 days. The ability of the cells to invade through reconstituted basement membrane (Matrigel) and to establish experimental pulmonary metastatic foci in C57BL/6 mice decreased after genistein exposure. The genistein-treated cells were also prevented from attaching to Matrigel and spread extremely poorly on the ECM substratum. Immunoblot analysis showed that tyrosine phosphorylation of a 125-kD protein in response to cell spreading on Matrigel was suppressed in the genistein-treated cells. Adhesion-induced protein tyrosine phosphorylation represents the earlier and specific event in the activation of ECM signaling, so this result implied ECM signaling was impaired in the treated cells. With immunofluorescence microscopy, the adhesion-induced tyrosine phosphorylated proteins were located at the pericytoplasms of well-spread cells, but not at the periphery of poorly spread genistein-treated cells. Therefore, this paper suggests that genistein might impair ECM signaling and subsequently prevent cancer cells from spreading well and invading or establishing metastasis through the suppression of adhesion-induced protein tyrosine phosphorylation. PTKs and adhesion-induced protein tyrosine phosphorylation might play a role in the control of invasion and metastasis.

  12. Tyrosine Kinase Inhibition in HPV-related Squamous Cell Carcinoma Reveals Beneficial Expression of cKIT and Src.

    Science.gov (United States)

    Kramer, Benedikt; Kneissle, Marcel; Birk, Richard; Rotter, Nicole; Aderhold, Christoph

    2018-05-01

    Therapeutic options of locally advanced or metastatic head and neck squamous cell carcinoma (HNSCC) are limited. Src and cKIT are key protein regulators for local tumor progression. The aim of the study was to investigate the therapeutic potential of targeted therapies in human squamous cell carcinoma (HNSCC) in vitro. Therefore, the influence of the selective tyrosine kinase inhibitors niotinib, dasatinib, erlotinib, gefitinib and afatinib on Src and cKIT expression in Human papilloma virus (HPV)-positive and HPV-negative squamous cancer cells (SCC) was analyzed in vitro. ELISA was performed to evaluate the expression of Src and cKIT under the influence of nilotinib, dasatinib, erlotinib, gefitinib and afatinib (10 μmol/l) in HPV-negative and HPV-positive SCC (24-96 h of incubation). Gefitinib significantly increased cKIT expression in HPV-positive and HPV-negative cells whereas nilotinib and afatinib decreased cKIT expression in HPV-positive SCC. The influence of tyrosine kinase inhibitors in HPV-negative SCC was marginal. Surprisingly, Src expression was significantly increased by all tested tyrosine kinase inhibitors in HPV-positive SCC. The results revealed beneficial and unexpected information concerning the interaction of selective tyrosine kinase inhibitors and the tumor biology of HNSCC. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  13. 123I-Iodomethyl tyrosine radiochemical synthesis and quantification of residual impurities after SepPak purification

    International Nuclear Information System (INIS)

    Matte, G.; Abrams, D.; Kumar, P.; Mercer, J.

    2002-01-01

    [123-I]-Iodomethyl tyrosine, an analog of tyrosine, is used as a radiopharmaceutical to detect malignant tissue in vivo. Initial synthesis report removal of the starting material using HPLC reversed phase chromatography as well as a simple method using a C18-SepPak cartridge when an HPLC system is not available. Small amounts of residual starting material have not been reported to interfere with tumor uptake following biodistribution in vivo. However, in vitro tissue culture studies do require the final product to be free of un-reacted methyl tyrosine. Our goal was to quantify the amount of residual methyl tyrosine after C18-SepPak purification to confirm that 123 I methyl tyrosine purified in this manner would be suitable tissue culture studies. A preconditioned (rinsed with 2 ml Ethanol and 8 ml PBS) C18-SepPak cartridge is loaded with the 123 I-iodomethyl tyrosine reaction mixture and washed with 8 mL of PBS to remove the un-reacted methyl tyrosine and free 123 I-iodide. The cartridge is then eluted with a 20% alcohol/PBS mixture to recover the 123 I-iodomethyl tyrosine. Paper chromatography confirmed the removal of un-reacted 123 I-iodide. A parallel study with a methyl tyrosine standard was used to confirm the removal of the methyl tyrosine from the SepPak cartridge during the washing with 8 mL of PBS. Fractions were collected and UV absorbance was recorded. A standard curve was prepared using the UV absorbance of serial dilutions of methyl tyrosine. The detection limit was in the order of ng/mL. An elution profile of both 123 I methyl tyrosine and methyl tyrosine was obtained and shows that traces of methyl tyrosine can still be present after an 8 mL PBS wash

  14. Subcutaneous L-tyrosine elicits cutaneous analgesia in response to local skin pinprick in rats.

    Science.gov (United States)

    Hung, Ching-Hsia; Chiu, Chong-Chi; Liu, Kuo-Sheng; Chen, Yu-Wen; Wang, Jhi-Joung

    2015-10-15

    The purpose of the study was to estimate the ability of L-tyrosine to induce cutaneous analgesia and to investigate the interaction between L-tyrosine and the local anesthetic lidocaine. After subcutaneously injecting the rats with L-tyrosine and lidocaine in a dose-dependent manner, cutaneous analgesia (by blocking the cutaneous trunci muscle reflex-CTMR) was evaluated in response to the local pinprick. The drug-drug interaction was analyzed by using an isobolographic method. We showed that both L-tyrosine and lidocaine produced dose-dependent cutaneous analgesia. On the 50% effective dose (ED50) basis, the rank of drug potency was lidocaine (5.09 [4.88-5.38] μmol)>L-tyrosine (39.1 [36.5-41.8] μmol) (Ptyrosine lasted longer than that caused by lidocaine (Ptyrosine exhibited an additive effect on infiltrative cutaneous analgesia. Our pre-clinical study demonstrated that L-tyrosine elicits the local/cutaneous analgesia, and the interaction between L-tyrosine and lidocaine is additive. L-tyrosine has a lower potency but much greater duration of cutaneous analgesia than lidocaine. Adding L-tyrosine to lidocaine preparations showed greater duration of cutaneous analgesia compared with lidocaine alone. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Tyrosine phosphorylation of Kv1.5 is upregulated in intrauterine growth retardation rats with exaggerated pulmonary hypertension

    Directory of Open Access Journals (Sweden)

    L.C. Fu

    2017-09-01

    Full Text Available Intrauterine growth retardation (IUGR is associated with the development of adult-onset diseases, including pulmonary hypertension. However, the underlying mechanism of the early nutritional insult that results in pulmonary vascular dysfunction later in life is not fully understood. Here, we investigated the role of tyrosine phosphorylation of voltage-gated potassium channel 1.5 (Kv1.5 in this prenatal event that results in exaggerated adult vascular dysfunction. A rat model of chronic hypoxia (2 weeks of hypoxia at 12 weeks old following IUGR was used to investigate the physiological and structural effect of intrauterine malnutrition on the pulmonary artery by evaluating pulmonary artery systolic pressure and vascular diameter in male rats. Kv1.5 expression and tyrosine phosphorylation in pulmonary artery smooth muscle cells (PASMCs were determined. We found that IUGR increased mean pulmonary artery pressure and resulted in thicker pulmonary artery smooth muscle layer in 14-week-old rats after 2 weeks of hypoxia, while no difference was observed in normoxia groups. In the PASMCs of IUGR-hypoxia rats, Kv1.5 mRNA and protein expression decreased while that of tyrosine-phosphorylated Kv1.5 significantly increased. These results demonstrate that IUGR leads to exaggerated chronic hypoxia pulmonary arterial hypertension (CH-PAH in association with decreased Kv1.5 expression in PASMCs. This phenomenon may be mediated by increased tyrosine phosphorylation of Kv1.5 in PASMCs and it provides new insight into the prevention and treatment of IUGR-related CH-PAH.

  16. Protein Tyrosine Nitration: Biochemical Mechanisms and Structural Basis of its Functional Effects

    Science.gov (United States)

    Radi, Rafael

    2012-01-01

    CONSPECTUS The nitration of protein tyrosine residues to 3-nitrotyrosine represents an oxidative postranslational modification that unveils the disruption of nitric oxide (•NO) signaling and metabolism towards pro-oxidant processes. Indeed, excess levels of reactive oxygen species in the presence of •NO or •NO-derived metabolites lead to the formation of nitrating species such as peroxynitrite. Thus, protein 3-nitrotyrosine has been established as a biomarker of cell, tissue and systemic “nitroxidative stress”. Moreover, tyrosine nitration modifies key properties of the amino acid (i.e. phenol group pKa, redox potential, hydrophobicity and volume). Thus, the incorporation of a nitro group (−NO2) to protein tyrosines can lead to profound structural and functional changes, some of which contribute to altered cell and tissue homeostasis. In this Account, I describe our current efforts to define 1) biologically-relevant mechanisms of protein tyrosine nitration and 2) how this modification can cause changes in protein structure and function at the molecular level. First, the relevance of protein tyrosine nitration via free radical-mediated reactions (in both peroxynitrite-dependent or independent pathways) involving the intermediacy of tyrosyl radical (Tyr•) will be underscored. This feature of the nitration process becomes critical as Tyr• can take variable fates, including the formation of 3-nitrotyrosine. Fast kinetic techniques, electron paramagnetic resonance (EPR) studies, bioanalytical methods and kinetic simulations have altogether assisted to characterize and fingerprint the reactions of tyrosine with peroxynitrite and one-electron oxidants and its further evolution to 3-nitrotyrosine. Recent findings show that nitration of tyrosines in proteins associated to biomembranes is linked to the lipid peroxidation process via a connecting reaction that involves the one-electron oxidation of tyrosine by lipid peroxyl radicals (LOO•). Second

  17. Fourier transform infrared difference spectroscopy of bacteriorhodopsin and its photoproducts regenerated with deuterated tyrosine

    International Nuclear Information System (INIS)

    Dollinger, G.; Eisenstein, L.; Lin, S.L.; Nakanishi, K.; Termini, J.

    1986-01-01

    Fourier transform infrared (FTIR) difference spectroscopy has been used to detect the vibrational modes due to tyrosine residues in the protein that change in position or intensity between light-adapted bacteriorhodopsin (LA) and other species, namely, the K and M intermediates and dark-adapted bacteriorhodopsin (DA). To aid in the identification of the bands that change in these various species, the FTIR spectra of the free amino acids Tyr-d0, Tyr-d2 ( 2 H at positions ortho to OH), and Tyr-d4 ( 2 H at positions ortho and meta to OH) were measured in H 2 O and D 2 O at low and high pH. The characteristic frequencies of the Tyr species obtained in this manner were then used to identify the changes in protonation state of the tyrosine residues in the various bacteriorhodopsin species. The two diagnostically most useful bands were the approximately 1480-cm-1 band of Tyr(OH)-d2 and the approximately 1277-cm-1 band of Tyr(O-)-d0. Mainly by observing the appearance or disappearance of these bands in the difference spectra of pigments incorporating the tyrosine isotopes, it was possible to identify the following: in LA, one tyrosine and one tyrosinate; in the K intermediate, two tyrosines; in the M intermediate, one tyrosine and one tyrosinate; and in DA, two tyrosines. Since these residues were observed in the difference spectra K/LA, M/LA, and DA/LA, they represent the tyrosine or tyrosinate groups that most likely undergo changes in protonation state due to the conversions. These changes are most likely linked to the proton translocation process of bacteriorhodopsin

  18. Rates and energetics of tyrosine ring flips in yeast iso-2-cytochrome c

    International Nuclear Information System (INIS)

    Nall, B.T.; Zuniga, E.H.

    1990-01-01

    Isotope-edited nuclear magnetic resonance spectroscopy is used to monitor ring flip motion of the five tyrosine side chains in the oxidized and reduced forms of yeast iso-2-cytochrome c. With specifically labeled protein purified from yeast grown on media containing [3,5- 13 C]tyrosine, isotope-edited one-dimensional proton spectra have been collected over a 5-55 degree C temperature range. The spectra allow selective observation of the 10 3,5 tyrosine ring proton resonances and, using a two-site exchange model, allow estimation of the temperature dependence of ring flip rates from motion-induced changes in proton line shapes. For the reduced protein, tyrosines II and IV are in fast exchange throughout the temperature range investigated, or lack resolvable differences in static chemical shifts for the 3,5 ring protons. Tyrosines I, III, and V are in sloe exchange at low temperatures and in fast exchange at high temperatures. Spectral simulations give flip rates for individual tyrosines in a range of one flip per second at low temperatures to thousands of flips per second at high temperatures. Eyring plots show that two of the tyrosines (I and III) have essentially the same activation parameters. Tentative sequence-specific assignments for the tyrosines in reduced iso-2 are suggested by comparison to horse cytochrome c. For oxidized iso-2, five resonances are observed at high temperatures, suggesting flip rates for all five tyrosines sufficient to average static chemical shift differences. At lower temperatures, there is evidence of intermediate and slow flipping for some of the rings

  19. Mechanism-based inactivation of cytochrome P-450 dependent benzo[a]pyrene hydroxylase activity by acetylenic and olefinic polycyclic arylhydrocarbons

    International Nuclear Information System (INIS)

    Gan, L.S.

    1986-01-01

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

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

    Science.gov (United States)

    Nilsson, G E; Tottmar, O

    1987-04-21

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

  1. Cortactin Tyrosine Phosphorylation Promotes Its Deacetylation and Inhibits Cell Spreading

    Science.gov (United States)

    Meiler, Eugenia; Nieto-Pelegrín, Elvira; Martinez-Quiles, Narcisa

    2012-01-01

    Background Cortactin is a classical Src kinase substrate that participates in actin cytoskeletal dynamics by activating the Arp2/3 complex and interacting with other regulatory proteins, including FAK. Cortactin has various domains that may contribute to the assembly of different protein platforms to achieve process specificity. Though the protein is known to be regulated by post-translational modifications such as phosphorylation and acetylation, how tyrosine phosphorylation regulates cortactin activity is poorly understood. Since the basal level of tyrosine phosphorylation is low, this question must be studied using stimulated cell cultures, which are physiologically relevant but unreliable and difficult to work with. In fact, their unreliability may be the cause of some contradictory findings about the dynamics of tyrosine phosphorylation of cortactin in different processes. Methodology/Principal Findings In the present study, we try to overcome these problems by using a Functional Interaction Trap (FIT) system, which involves cotransfecting cells with a kinase (Src) and a target protein (cortactin), both of which are fused to complementary leucine-zipper domains. The FIT system allowed us to control precisely the tyrosine phosphorylation of cortactin and explore its relationship with cortactin acetylation. Conclusions/Significance Using this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading. We confirmed the results from the FIT system by examining endogenous cortactin in different cell types. Furthermore, we demonstrate that cell spreading promotes the association of cortactin and FAK and that tyrosine phosphorylation of cortactin disrupts this interaction, which may explain how it inhibits cell spreading. PMID:22479425

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

    Science.gov (United States)

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

    1976-09-10

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

  3. Parkinsonism in phenylketonuria: a consequence of dopamine depletion?

    NARCIS (Netherlands)

    Velema, Marieke; Boot, Erik; Engelen, Marc; Hollak, Carla

    2015-01-01

    Phenylketonuria (PKU) is caused by a deficiency or inactivity of the enzyme phenylalanine hydroxylase that converts phenylalanine (Phe) to tyrosine (Tyr). It has been proposed that a reduction of brain Tyr levels, as well as reduced activity of the key regulatory enzyme of dopamine (DA) synthesis

  4. 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

  5. Tyrosine isomers mediate the classical phenomenon of concomitant tumor resistance.

    Science.gov (United States)

    Ruggiero, Raúl A; Bruzzo, Juan; Chiarella, Paula; di Gianni, Pedro; Isturiz, Martín A; Linskens, Susana; Speziale, Norma; Meiss, Roberto P; Bustuoabad, Oscar D; Pasqualini, Christiane D

    2011-11-15

    Concomitant tumor resistance (CR) is a phenomenon originally described in 1906 in which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis. Although recent studies have indicated that T-cell-dependent processes mediate CR in hosts bearing immunogenic small tumors, manifestations of CR induced by immunogenic and nonimmunogenic large tumors have been associated with an elusive serum factor. In this study, we identify this serum factor as tyrosine in its meta and ortho isoforms. In three different murine models of cancer that generate CR, both meta-tyrosine and ortho-tyrosine inhibited tumor growth. In addition, we showed that both isoforms of tyrosine blocked metastasis in a fourth model that does not generate CR but is sensitive to CR induced by other tumors. Mechanistic studies showed that the antitumor effects of the tyrosine isoforms were mediated, in part, by early inhibition of mitogen-activated protein/extracellular signal-regulated kinase pathway and inactivation of STAT3, potentially driving tumor cells into a state of dormancy. By revealing a molecular basis for the classical phenomenon of CR, our findings may stimulate new generalized approaches to limit the development of metastases that arise after resection of primary tumors, an issue of pivotal importance to oncologists and their patients. ©2011 AACR

  6. Carotenoid β-Ring Hydroxylase and Ketolase from Marine Bacteria—Promiscuous Enzymes for Synthesizing Functional Xanthophylls

    Science.gov (United States)

    Misawa, Norihiko

    2011-01-01

    Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C40-type dicyclic carotenoids containing two β-end groups (β rings) that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4′)-ketolase (4(4′)-oxygenase; CrtW) and hydroxylated by carotenoid β-ring 3(3′)-hydroxylase (CrtZ). In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2′)-hydroxylase (CrtG). This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids) in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s)-2(2′)-hydroxylated carotenoids). PMID:21673887

  7. Carotenoid β-Ring Hydroxylase and Ketolase from Marine Bacteria—Promiscuous Enzymes for Synthesizing Functional Xanthophylls

    Directory of Open Access Journals (Sweden)

    Norihiko Misawa

    2011-05-01

    Full Text Available Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C40-type dicyclic carotenoids containing two β-end groups (β rings that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4′-ketolase (4(4′-oxygenase; CrtW and hydroxylated by carotenoid β-ring 3(3′-hydroxylase (CrtZ. In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2′-hydroxylase (CrtG. This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s-2(2′-hydroxylated carotenoids.

  8. Carotenoid β-ring hydroxylase and ketolase from marine bacteria-promiscuous enzymes for synthesizing functional xanthophylls.

    Science.gov (United States)

    Misawa, Norihiko

    2011-01-01

    Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C₄₀-type dicyclic carotenoids containing two β-end groups (β rings) that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4')-ketolase (4(4')-oxygenase; CrtW) and hydroxylated by carotenoid β-ring 3(3')-hydroxylase (CrtZ). In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2')-hydroxylase (CrtG). This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids) in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s)-2(2')-hydroxylated carotenoids).

  9. Tyrosine dephosphorylation enhances the therapeutic target activity of epidermal growth factor receptor (EGFR) by disrupting its interaction with estrogen receptor (ER).

    Science.gov (United States)

    Ma, Shao; Yin, Ning; Qi, Xiaomei; Pfister, Sandra L; Zhang, Mei-Jie; Ma, Rong; Chen, Guan

    2015-05-30

    Protein-protein interactions can increase or decrease its therapeutic target activity and the determining factors involved, however, are largely unknown. Here, we report that tyrosine-dephosphorylation of epidermal growth factor receptor (EGFR) increases its therapeutic target activity by disrupting its interaction with estrogen receptor (ER). Protein tyrosine phosphatase H1 (PTPH1) dephosphorylates the tyrosine kinase EGFR, disrupts its interaction with the nuclear receptor ER, and increases breast cancer sensitivity to small molecule tyrosine kinase inhibitors (TKIs). These effects require PTPH1 catalytic activity and its interaction with EGFR, suggesting that the phosphatase may increase the sensitivity by dephosphorylating EGFR leading to its dissociation with ER. Consistent with this notion, a nuclear-localization defective ER has a higher EGFR-binding activity and confers the resistance to TKI-induced growth inhibition. Additional analysis show that PTPH1 stabilizes EGFR, stimulates the membranous EGFR accumulation, and enhances the growth-inhibitory activity of a combination therapy of TKIs with an anti-estrogen. Since EGFR and ER both are substrates for PTPH1 in vitro and in intact cells, these results indicate that an inhibitory EGFR-ER protein complex can be switched off through a competitive enzyme-substrate binding. Our results would have important implications for the treatment of breast cancer with targeted therapeutics.

  10. Effect of tyrosine kinase blockade on norepinephrine-induced cytosolic calcium response in rat afferent arterioles

    DEFF Research Database (Denmark)

    Salomonsson, Max; Arendshorst, William J

    2004-01-01

    We used genistein (Gen) and tyrphostin 23 (Tyr-23) to evaluate the importance of tyrosine phosphorylation in norepinephrine (NE)-induced changes in intracellular free calcium concentration ([Ca(2+)](i)) in rat afferent arterioles. [Ca(2+)](i) was measured in microdissected arterioles using...... ratiometric photometry of fura 2 fluorescence. The control [Ca(2+)](i) response to NE (1 microM) consisted of a rapid initial peak followed by a plateau phase sustained above baseline. Pretreatment with the tyrosine kinase inhibitor Tyr-23 (50 microM, 10 min) caused a slow 40% increase in baseline [Ca(2+)](i...... of nifedipine and Tyr-23 were not additive. Nifedipine had no inhibitory effect after Tyr-23 pretreatment, indicating Tyr-23 inhibition of Ca(2+) entry. Another tyrosine kinase inhibitor, Gen (5 and 50 microM), did not affect baseline [Ca(2+)](i). High-dose Gen inhibited the peak and plateau response to NE...

  11. Novel deletion alleles carrying CYP21A1P/A2 chimeric genes in Brazilian patients with 21-hydroxylase deficiency

    Directory of Open Access Journals (Sweden)

    Guerra-Júnior Gil

    2010-06-01

    Full Text Available Abstract Background Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is caused by deletions, large gene conversions or mutations in CYP21A2 gene. The human gene is located at 6p21.3 within a locus containing the genes for putative serine/threonine Kinase RP, complement C4, steroid 21-hydroxylase CYP21 tenascin TNX, normally, in a duplicated cluster known as RCCX module. The CYP21 extra copy is a pseudogene (CYP21A1P. In Brazil, 30-kb deletion forming monomodular alleles that carry chimeric CYP21A1P/A2 genes corresponds to ~9% of disease-causing alleles. Such alleles are considered to result from unequal crossovers within the bimodular C4/CYP21 locus. Depending on the localization of recombination breakpoint, different alleles can be generated conferring the locus high degree of allelic variability. The purpose of the study was to investigate the variability of deleted alleles in patients with 21-hydroxylase deficiency. Methods We used different techniques to investigate the variability of 30-kb deletion alleles in patients with 21-hydroxylase deficiency. Alleles were first selected after Southern blotting. The composition of CYP21A1P/A2 chimeric genes was investigated by ASO-PCR and MLPA analyses followed by sequencing to refine the location of recombination breakpoints. Twenty patients carrying at least one allele with C4/CYP21 30-kb deletion were included in the study. Results An allele carrying a CYP21A1P/A2 chimeric gene was found unusually associated to a C4B/C4A Taq I 6.4-kb fragment, generally associated to C4B and CYP21A1P deletions. A novel haplotype bearing both p.P34L and p.H62L, novel and rare mutations, respectively, was identified in exon 1, however p.P30L, the most frequent pseudogene-derived mutation in this exon, was absent. Four unrelated patients showed this haplotype. Absence of p.P34L in CYP21A1P of normal controls indicated that it is not derived from pseudogene. In addition, the combination of different

  12. Intracellular Growth Is Dependent on Tyrosine Catabolism in the Dimorphic Fungal Pathogen Penicillium marneffei

    Science.gov (United States)

    Boyce, Kylie J.; McLauchlan, Alisha; Schreider, Lena; Andrianopoulos, Alex

    2015-01-01

    During infection, pathogens must utilise the available nutrient sources in order to grow while simultaneously evading or tolerating the host’s defence systems. Amino acids are an important nutritional source for pathogenic fungi and can be assimilated from host proteins to provide both carbon and nitrogen. The hpdA gene of the dimorphic fungus Penicillium marneffei, which encodes an enzyme which catalyses the second step of tyrosine catabolism, was identified as up-regulated in pathogenic yeast cells. As well as enabling the fungus to acquire carbon and nitrogen, tyrosine is also a precursor in the formation of two types of protective melanin; DOPA melanin and pyomelanin. Chemical inhibition of HpdA in P. marneffei inhibits ex vivo yeast cell production suggesting that tyrosine is a key nutrient source during infectious growth. The genes required for tyrosine catabolism, including hpdA, are located in a gene cluster and the expression of these genes is induced in the presence of tyrosine. A gene (hmgR) encoding a Zn(II)2-Cys6 binuclear cluster transcription factor is present within the cluster and is required for tyrosine induced expression and repression in the presence of a preferred nitrogen source. AreA, the GATA-type transcription factor which regulates the global response to limiting nitrogen conditions negatively regulates expression of cluster genes in the absence of tyrosine and is required for nitrogen metabolite repression. Deletion of the tyrosine catabolic genes in the cluster affects growth on tyrosine as either a nitrogen or carbon source and affects pyomelanin, but not DOPA melanin, production. In contrast to other genes of the tyrosine catabolic cluster, deletion of hpdA results in no growth within macrophages. This suggests that the ability to catabolise tyrosine is not required for macrophage infection and that HpdA has an additional novel role to that of tyrosine catabolism and pyomelanin production during growth in host cells. PMID:25812137

  13. 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.

  14. Sorbitol Can Fuel Mouse Sperm Motility and Protein Tyrosine Phosphorylation via Sorbitol Dehydrogenase1

    Science.gov (United States)

    Cao, Wenlei; Aghajanian, Haig K.; Haig-Ladewig, Lisa A.; Gerton, George L.

    2008-01-01

    Energy sources that can be metabolized to yield ATP are essential for normal sperm functions such as motility. Two major monosaccharides, sorbitol and fructose, are present in semen. Furthermore, sorbitol dehydrogenase (SORD) can convert sorbitol to fructose, which can then be metabolized via the glycolytic pathway in sperm to make ATP. Here we characterize Sord mRNA and SORD expression during mouse spermatogenesis and examine the ability of sorbitol to support epididymal sperm motility and tyrosine phosphorylation. Sord mRNA levels increased during the course of spermatogenic differentiation. SORD protein, however, was first detected at the condensing spermatid stage. By indirect immunofluorescence, SORD was present along the length of the flagella of caudal epididymal sperm. Furthermore, immunoelectron microscopy showed that SORD was associated with mitochondria and the plasma membranes of sperm. Sperm incubated with sorbitol maintained motility, indicating that sorbitol was utilized as an energy source. Sorbitol, as well as glucose and fructose, were not essential to induce hyperactive motility. Protein tyrosine phosphorylation increased in a similar manner when sorbitol was substituted for glucose in the incubation medium used for sperm capacitation. These results indicate that sorbitol can serve as an alternative energy source for sperm motility and protein tyrosine phosphorylation. PMID:18799757

  15. 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

  16. 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.

  17. 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...

  18. Uptake of Tyrosine Amino Acid on Nano-Graphene Oxide

    Directory of Open Access Journals (Sweden)

    Hossam M. Nassef

    2018-01-01

    Full Text Available Graphene oxide (GO is emerging as a promising nanomaterial with potential application in the detection and analysis of amino acids, DNA, enzymes, and proteins in biological fluid samples. So, the reaction of GO with amino acids should be characterized and determined before using it in biosensing methods and devices. In this study, the reaction of tyrosine amino acid (Tyr with GO was characterized using FT-IR, UV-vis spectrophotometry, and scanning electron microscopy (SEM before its use. The optimum conditions for GO’s interaction with Tyr amino acid have been studied under variable conditions. The optimum conditions of pH, temperature, shaking time, and GO and tyrosine concentrations for the uptaking of tyrosine amino acid onto the GO’s surface from aqueous solution were determined. The SEM analysis showed that the GO supplied was in a particle size range between 5.4 and 8.1 nm. A pH of 8.4–9.4 at 25 °C and 5 min of shaking time were the optimum conditions for a maximum uptake of 1.4 μg/mL of tyrosine amino acid onto 0.2 mg/mL of GO.

  19. Interactions between Type III receptor tyrosine phosphatases and growth factor receptor tyrosine kinases regulate tracheal tube formation in Drosophila

    Directory of Open Access Journals (Sweden)

    Mili Jeon

    2012-04-01

    The respiratory (tracheal system of the Drosophila melanogaster larva is an intricate branched network of air-filled tubes. Its developmental logic is similar in some ways to that of the vertebrate vascular system. We previously described a unique embryonic tracheal tubulogenesis phenotype caused by loss of both of the Type III receptor tyrosine phosphatases (RPTPs, Ptp4E and Ptp10D. In Ptp4E Ptp10D double mutants, the linear tubes in unicellular and terminal tracheal branches are converted into bubble-like cysts that incorporate apical cell surface markers. This tube geometry phenotype is modulated by changes in the activity or expression of the epidermal growth factor receptor (Egfr tyrosine kinase (TK. Ptp10D physically interacts with Egfr. Here we demonstrate that the Ptp4E Ptp10D phenotype is the consequence of the loss of negative regulation by the RPTPs of three growth factor receptor TKs: Egfr, Breathless and Pvr. Reducing the activity of any of the three kinases by tracheal expression of dominant-negative mutants suppresses cyst formation. By competing dominant-negative and constitutively active kinase mutants against each other, we show that the three RTKs have partially interchangeable activities, so that increasing the activity of one kinase can compensate for the effects of reducing the activity of another. This implies that SH2-domain downstream effectors that are required for the phenotype are likely to be able to interact with phosphotyrosine sites on all three receptor TKs. We also show that the phenotype involves increases in signaling through the MAP kinase and Rho GTPase pathways.

  20. Protein-bound tyrosine oxidation, nitration and chlorination by-products assessed by ultraperformance liquid chromatography coupled to tandem mass spectrometry.

    Science.gov (United States)

    Torres-Cuevas, Isabel; Kuligowski, Julia; Cárcel, María; Cháfer-Pericás, Consuelo; Asensi, Miguel; Solberg, Rønnaug; Cubells, Elena; Nuñez, Antonio; Saugstad, Ola Didrik; Vento, Máximo; Escobar, Javier

    2016-03-24

    Free radicals cause alterations in cellular protein structure and function. Oxidized, nitrated, and chlorinated modifications of aromatic amino acids including phenylalanine and tyrosine are reliable biomarkers of oxidative stress and inflammation in clinical conditions. To develop, validate and apply a rapid method for the quantification of known hallmarks of tyrosine oxidation, nitration and chlorination in plasma and tissue proteins providing a snapshot of the oxidative stress and inflammatory status of the organism and of target organs respectively. The extraction and clean up procedure entailed protein precipitation, followed by protein re-suspension and enzymatic digestion with pronase. An Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MS/MS) method was developed to quantify protein released ortho-tyrosine (o-Tyr), meta-tyrosine (m-Tyr), 3-nitrotyrosine (3NO2-Tyr) and 3-chlorotyrosine (3Cl-Tyr) as well as native phenylalanine (Phe) and tyrosine (p-Tyr) in plasma and tissue from a validated hypoxic newborn piglet experimental model. In plasma there was a significant increase in the 3NO2-Tyr/p-Tyr ratio. On the other hand m-Tyr/Phe and 3Cl-Tyr/p-Tyr ratios were significantly increased in liver of hypoxic compared with normoxic animals. Although no significant differences were found in brain tissue, a clear tendency to increased ratios was observed under hypoxic conditions. UPLC-MS/MS has proven suitable for the analysis of plasma and tissue samples from newborn piglets. The analysis of biomarkers of protein oxidation, nitration and chlorination will be applied in future studies aiming to provide a deeper insight into the mechanisms of oxidation-derived protein modification caused during neonatal asphyxia and resuscitation. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Fox-2 protein regulates the alternative splicing of scleroderma-associated lysyl hydroxylase 2 messenger RNA.

    Science.gov (United States)

    Seth, Puneet; Yeowell, Heather N

    2010-04-01

    Scleroderma (systemic sclerosis [SSc]) is a complex connective tissue disorder characterized by hardening and thickening of the skin. One hallmark of scleroderma is excessive accumulation of collagen accompanied by increased levels of pyridinoline collagen crosslinks derived from hydroxylysine residues in the collagen telopeptide domains. Lysyl hydroxylase 2 (LH2), an important alternatively spliced enzyme in collagen biosynthesis, acts as a collagen telopeptide hydroxylase. Changes in the pattern of LH2 alternative splicing, favoring increased inclusion of the alternatively spliced LH2 exon 13A, thereby increasing the levels of the long transcript of LH2 (LH2[long]), are linked to scleroderma disease. This study was undertaken to examine the role played by RNA binding protein Fox-2 in regulating exon 13A inclusion, which leads to the generation of scleroderma-associated LH2(long) messenger RNA (mRNA). Phylogenetic sequence analysis of introns flanking exon 13A was performed. A tetracycline-inducible system in T-Rex 293 cells was used to induce Fox-2 protein, and endogenous LH2(long) mRNA was determined by reverse transcriptase-polymerase chain reaction. An LH2 minigene was designed, validated, and used in Fox-2 overexpression and mutagenesis experiments. Knockdown of Fox-2 was performed in mouse embryonic fibroblasts and in fibroblasts from SSc patients. Overexpression of Fox-2 enhanced the inclusion of exon 13A and increased the generation of LH2(long) mRNA, whereas knockdown of Fox-2 decreased LH2(long) transcripts. Mutational analysis of an LH2 minigene demonstrated that 2 of the 4 Fox binding motifs flanking LH2 exon 13A are required for inclusion of exon 13A. In early passage fibroblasts derived from patients with scleroderma, the knockdown of Fox-2 protein significantly decreased the endogenous levels of LH2(long) mRNA. Our findings indicate that Fox-2 plays an integral role in the regulation of LH2 splicing. Knockdown of Fox-2 and other methods to decrease

  2. Effect of nerve activity on transport of nerve growth factor and dopamine β-hydroxylase antibodies in sympathetic neurones

    International Nuclear Information System (INIS)

    Lees, G.; Chubb, I.; Freeman, C.; Geffen, L.; Rush, R.

    1981-01-01

    The effect of nerve activity on the uptake and retrograde transport of nerve growth factor (NGF) and dopamine β-hydroxylase (DBH) antibodies was studied by injecting 125 I-labelled NGF and anti-DBH into the anterior eye chamber of guinea-pigs. Decentralization of the ipsilateral superior cervical ganglion (SCG) had no significant effect on the retrograde transport of either NGF or anti-DBH. Phenoxybenzamine produced a 50% increase in anti-DBH but not NGF accumulation and this effect was prevented by prior decentralization. This demonstrates that NGF is taken up independently of the retrieval of synaptic vesicle components. (Auth.)

  3. DETECTION AND PURIFICATION OF TYROSINE-SULFATED PROTEINS USING A NOVEL ANTI-SULFOTYROSINE MONOCLONAL ANTIBODY*

    Science.gov (United States)

    Hoffhines, Adam J.; Damoc, Eugen; Bridges, Kristie G.; Leary, Julie A.; Moore, Kevin L.

    2006-01-01

    Protein-tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 & TPST-2) that catalyze the transfer of sulfate to tyrosine residues in secreted and transmembrane proteins. Tyrosine sulfation plays a role in protein-protein interactions in several well-defined systems. Although dozens of tyrosine-sulfated proteins are known, many more are likely to exist and await description. Advancing our understanding of the importance of tyrosine sulfation in biological systems requires the development of new tools for the detection and study of tyrosine-sulfated proteins. We have developed a novel anti-sulfotyrosine monoclonal antibody, called PSG2, that binds with high affinity and exquisite specificity to sulfotyrosine residues in peptides and proteins independent of sequence context. We show that it can detect tyrosinesulfated proteins in complex biological samples and can be used as a probe to assess the role of tyrosine sulfation in protein function. We also demonstrate the utility of PSG2 in the purification of tyrosine-sulfated proteins from crude tissue samples. Finally, Western blot analysis using PSG2 indicates that certain sperm/epididymal proteins are undersulfated in Tpst2−/− mice. This indicates that TPST-1 and TPST-2 have distinct macromolecular substrate specificities and provides clues as to the molecular mechanism of the infertility of Tpst2−/− males. PSG2 should be widely applicable for identification of tyrosine-sulfated proteins in other systems and organisms. PMID:17046811

  4. Pre-Clinical Testing of New Hydroxybutyrate Analogues

    Science.gov (United States)

    2012-07-01

    will be assessed. Mac- 1 and GFAP immunostaining will be done to gauge inflammation as it relates to HDAC activity. Tyrosine hydroxylase and Nissl ... stained neurons will be counted and complex II histochemistry will be performed. Part II, we will prepare a new set of implanted mice treated with

  5. Preserved dopaminergic homeostasis and dopamine-related behaviour in hemizygous TH-Cre mice

    DEFF Research Database (Denmark)

    Thomsen, Annika Højrup Runegaard; Jensen, Kathrine L; Fitzpatrick, Ciarán M

    2017-01-01

    assessment of the dopaminergic system in hemizygous tyrosine hydroxylase (TH)-Cre mice in comparison to wild-type (WT) controls. Our data show that TH-Cre mice display preserved dopaminergic homeostasis with unaltered levels of TH and dopamine as well as unaffected dopamine turnover in striatum. TH-Cre mice...

  6. Site specific interaction between ZnO nanoparticles and tyrosine: A density functional theory study

    Science.gov (United States)

    Singh, Satvinder; Singh, Janpreet; Singh, Baljinder; Singh, Gurinder; Kaura, Aman; Tripathi, S. K.

    2018-05-01

    First Principles Calculations have been performed on ZnO/Tyrosine atomic complex to study site specific interaction of Tyrosine and ZnO nanoparticles. Calculated results shows that -COOH group present in Tyrosine is energetically more favorable than -NH2 group. Interactions show ionic bonding between ZnO and Tyrosine. All the calculations have been performed under the Density Functional Theory (DFT) framework. Structural and electronic properties of (ZnO)3/Tyrosine complex have been studied. Gaussian basis set approach has been adopted for the calculations. A ring type most stable (ZnO)3 atomic cluster has been modeled, analyzed and used for the calculations.

  7. Facile, one-step controlled synthesis of Se nanocrystals in the presence of L-tyrosine

    International Nuclear Information System (INIS)

    Wang Xiufang; Zhang Weiqiang; Shen Yuhua; Xie Anjian; Huang Lachun

    2011-01-01

    Highlights: → Se was synthesized via a novel and facile pathway. → The morphologies and the crystalline phases of Se can be easily controlled. → Tyrosine is excellent reducing agent and soft template for the synthesis of Se. → It is possible to provide an excellent route to obtain nanomaterials. - Abstract: Se with different morphologies was synthesized using L-tyrosine as reducing agent and soft template by means of hydrothermal method. The method was simple and convenient to handle. The reaction process was monitored using ultraviolet-visible spectroscopy (UV-vis) and Fourier-transform infrared spectroscopy (FTIR). The morphology and crystalline phase were determined by transmission electron microscopy (TEM), scanning electronic microscopy (SEM) and X-ray diffraction (XRD) pattern. The results show that the morphologies and the crystalline phases of Se can be easily controlled. By varying the concentration ratio of L-tyrosine to selenious acid, the morphologies and crystalline phases of Se were not changed, but the diameter of Se was different. Se nano-rods are obtained in the lower temperature, and there is a transformation of microspheres into nanorods of Se with the increase of reaction time or reaction temperature. In addition, we discuss the possible mechanism of the reduction of SeO 3 2- ions by L-tyrosine. The eco-friendly, biogenic synthesis strategy could be widely used for preparing inorganic/organic biocomposites.

  8. Solubilization and characterization of a novel tyrosine kinase from rat adipocytes

    International Nuclear Information System (INIS)

    Yagaloff, K.A.; Czech, M.P.

    1987-01-01

    The authors report the efficient solubilization and characterization of a Triton X-100 insoluble tyrosine kinase from rat adipocytes. Plasma membranes were prepared from rat epididymal fat pads and were solubilized in 1% Triton X-100. Following centrifugation, the pellet was solubilized for 15 min at 4 0 C using both ionic and non-ionic detergents. Tyrosine kinase activity was measured in the soluble and particulate fractions using the exogenous substrate poly(glu-tyr) in a TCA precipitation assay. Reactions were performed in 50mM Hepes, 10mM MgCl 2 and 100μM gamma[ 32 P]-ATP (10Ci/mmol) at 4 0 C with or without 1mg/ml of the polyaminoacid. Incorporation rates of 100 to 1000 pmol/min/mg were obtained, while endogenous [ 32 P] incorporation was typically less than 10% of that in the presence of poly(glu-tyr). More than 75% of the tyrosine kinase activity was recovered in the soluble supernatant using this assay methodology. The solubilized tyrosine kinase was found to require Mg 2+ or Mn 2+ but preferred Mg 2+ and was inhibited by high levels of Mn 2+ . Kinase activity was strongly inhibited by Ca 2+ (>50% at 1mM), NaCl (>50% at 250mM) and NH 4 SO 4 (>50% at 50mM) but was activated by 10μM heparin and 5mM dithiothreitol. These properties distinguish the solubilized tyrosine kinase from other cellular tyrosine kinases

  9. Enzymatic-induced upconversion photoinduced electron transfer for sensing tyrosine in human serum.

    Science.gov (United States)

    Wu, Qiongqiong; Fang, Aijin; Li, Haitao; Zhang, Youyu; Yao, Shouzhuo

    2016-03-15

    This paper reports a novel nanosensor for tyrosine based on photoinduced electron-transfer (PET) between NaYF4:Yb, Tm upconversion nanoparticles (UCNPs) and melanin-like polymers. Melanin-like films were obtained from catalytic oxidation of tyrosine by tyrosinase, and deposited on the surface of UCNPs, and then quenched the fluorescence of UCNPs. Under the optimized conditions, the fluorescence quenching of UCNPs showed a good linear response to tyrosine concentration in the range of 0.8-100 μΜ with a detection limit of 1.1 μΜ. Meanwhile, it showed good sensitivity, stability and has been successfully applied to the detection of tyrosine in human serum. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Topoisomerase I tyrosine phosphorylation site and the DNA-interactive site

    International Nuclear Information System (INIS)

    Roll, D.; Durban, E.

    1986-01-01

    Phosphorylation of topoisomerase I (topo I) at serine by NII kinase is accompanied by stimulation of enzymatic activity. In contrast, phosphorylation at tyrosine by tyrosine kinase seems to inhibit enzymatic activity. This inhibition may be caused by interference of the phosphorylated tyrosine residue with the interaction of topo I with DNA. To test this, topo I was labeled with crude membrane fraction enriched for EGF-receptor kinase in presence of γ-P32-ATP and electrophoresed on SDS-polyacrylamide gels. Stained topo I bands were excised, dried, digested with trypsin and analyzed on a C18 reverse-phase HPLC column. One major peak of radioactivity eluted at fraction 23 with 20% acetonitrile. To obtain the DNA-interactive site, topo I was incubated with pBR322 DNA labeled by nick-translation followed by DNase I treatment, and electrophoresis on SDS-polyacrylamide gels. Tryptic peptides were generated and analyzed by reverse-phase HPLC. A major peak of radioactivity eluted at fraction 16-18 with 15.5-17% acetonitrile. Studies are in progress to resolve whether (a) the two peptides are different, i.e. the tyrosine-P site and DNA-tyrosine interactive site are localized at different regions of the topo I or (b) the peptide sequences are identical but the covalent attachment of deoxynucleotides altered the peptide's elution from the HPLC column

  11. Expression Profiling of Tyrosine Kinase Genes

    National Research Council Canada - National Science Library

    Weier, Heinz

    2000-01-01

    ... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

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

    Science.gov (United States)

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

    1992-04-01

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

  13. Deficient tyrosine phosphorylation of c-Cbl and associated proteins in phorbol ester-resistant EL4 mouse thymoma cells.

    Science.gov (United States)

    Luo, X; Sando, J J

    1997-05-02

    Two tyrosine phosphoproteins in phorbol ester-sensitive EL4 (S-EL4) mouse thymoma cells have been identified as the p120 c-Cbl protooncogene product and the p85 subunit of phosphatidylinositol 3-kinase. Tyrosine phosphorylation of p120 and p85 increased rapidly after phorbol ester stimulation. Phorbol ester-resistant EL4 (R-EL4) cells expressed comparable amounts of c-Cbl and phosphatidylinositol 3-kinase protein but greatly diminished tyrosine phosphorylation. Co-immunoprecipitation experiments revealed complexes of c-Cbl with p85, and of p85 with the tyrosine kinase Lck in phorbol ester-stimulated S-EL4 but not in unstimulated S-EL4 or in R-EL4 cells. In vitro binding of c-Cbl with Lck SH2 or SH3 domains was detected in both S-EL4 and R-EL4 cells, suggesting that c-Cbl, p85, and Lck may form a ternary complex. In vitro kinase assays revealed phosphorylation of p85 by Lck only in phorbol ester-stimulated S-EL4 cells. Collectively, these results suggest that Cbl-p85 and Lck-p85 complexes may form in unstimulated S-EL4 and R-EL4 cells but were not detected due to absence of tyrosine phosphorylation of p85. Greatly decreased tyrosine phosphorylation of c-Cbl and p85 in the complexes may contribute to the failure of R-EL4 cells to respond to phorbol ester.

  14. Background levels and radiation dose yield of o-tyrosine in chicken meat

    International Nuclear Information System (INIS)

    Chuaqui-Offermanns, N.; McDougall, T.

    1991-01-01

    The measurement of o-tyrosine levels in poultry meat is a potential method for postirradiation dosimetry of poultry. The validity of using o-tyrosine for this purpose has not yet been established. As part of the validation process, the o-tyrosine content in unirradiated chicken meat, the radiation dose response curve, and the effects of postirradiation storage on o-tyrosine levels are examined. In 18 individual samples, the mean background level of o-tyrosine was 0.18 +/- 0.11 ppm (wet weight, 70% moisture), and the most frequent background level (60% of the cases) was between 0.05 and 0.15 ppm (wet weight, 70% moisture). In pooled samples of 10 chickens, the mean background level was 0.12 +/- 0.03 ppm (wet weight, 70% moisture). The levels were not significantly affected by storage at 5 degrees C (7 d) or by freezing the sample. The radiation dose response curve was linear within the dose range studied (0 to 10 kGy), with a slope of 0.127 + 0.003 ppm (wet weight)/kGy. Although there was some variation in the intercept (0.132 + 0.013), the slope was the same in all samples tested. Postirradiation storage at either 4 or 8 degrees C until spoilage did not affect the levels of o-tyrosine. These data indicate that o-tyrosine level may be useful for determining the absorbed dose in chicken meat gamma-irradiated to doses greater than 0.6 kGy. Further validation studies are continuing

  15. Acute oral administration of low doses of methylphenidate targets calretinin neurons in the rat septal area.

    Directory of Open Access Journals (Sweden)

    Alvaro eGarcía-Aviles

    2015-03-01

    Full Text Available Methylphenidate (MPD is a commonly administered drug to treat children suffering from attention deficit hyperactivity disorder (ADHD. Alterations in septal driven hippocampal theta rhythm may underlie attention deficits observed in these patients. Amongst others, the septo-hippocampal connections have long been acknowledged to be important in preserving hippocampal function. Thus, we wanted to ascertain if methylphenidate administration, which improves attention in patients, could affect septal areas connecting with hippocampus. We used low and orally administered methylphenidate doses (1.3; 2.7 and 5mg/Kg to rats what mimics the dosage range in humans. In our model, we observed no effect when using 1.3mg/Kg methylphenidate; whereas 2.7 and 5 mg/Kg induced a significant increase in c-fos expression specifically in the medial septum, an area intimately connected to the hippocampus. We analyzed dopaminergic areas such as nucleus accumbens and striatum, and found that only 5mg/Kg induced c-fos levels increase. In these areas tyrosine hydroxylase correlated well with c-fos staining, whereas in the medial septum the sparse tyrosine hydroxylase fibres did not overlap with c-fos positive neurons. Double immunofluorescence of c-fos with neuronal markers in the septal area revealed that co-localization with choline acethyl transferase, parvalbumin, and calbindin with c-fos did not change with MPD treatment; whereas, calretinin and c-fos double labeled neurons increased after MPD administration. Altogether, these results suggest that low and acute doses of methylphenidate primary target specific populations of caltretinin medial septal neurons.

  16. Synthesis of 2-[18F]fluoro-L-tyrosine via regiospecific fluoro-de-stannylation

    International Nuclear Information System (INIS)

    Hess, E.; Sichler, S.; Kluge, A.; Coenen, H.H.

    2002-01-01

    2-[ 18 F]Fluoro-L-tyrosine is a fluorine labelled amino acid, known to be incorporated into newly synthesised proteins, rendering it a potentially suitable tracer to image protein metabolism in vivo using positron emission tomography. For the electrophilic preparation of 2-[ 18 F]fluoro-L-tyrosine three protected 2-trialkylstannyl tyrosine derivatives have been synthesised for the first time as precursors. While O,N-di-Boc-2-triethylstannyl-L-tyrosine ethylester has proved to be suitable as precursor for radiosynthesis, imidazolidinon-derivatives of 2-triaklylstannyl tyrosine have not because of difficult fast hydrolysis of a phenolic O-methyl protective group. The di-Boc-tin derivative of tyrosine ethylester readily reacted with [ 18 F]F 2 , which was prepared via the 18 O(p,n) 18 F nuclear reaction. 2-[ 18 F]Fluoro-L-tyrosine was isolated after full deprotection with aqueous hydrobromic acid and HPLC purification with activities of 1.41±0.32 GBq. The isomeric and enantiomeric purity is high (both >99%). The preparation procedure is facile and easy to automate. The chemical yields of this fluoro-de-stannylation reaction as well as of the synthesis of 6-[ 18 F]fluoro-L-dopa, determined with an analogous precursor and non-radioactive fluorine under identical conditions, amounted to 42.7±1.6% and 60.2±2.8%, respectively

  17. Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

    Science.gov (United States)

    Takayama, S; White, M F; Kahn, C R

    1988-03-05

    The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function

  18. l-Tyrosine Contained in Dietary Supplement by Chemiluminescence Reaction of an Iron-Phthalocyanine Complex

    Directory of Open Access Journals (Sweden)

    Takao Ohtomo

    2012-01-01

    Full Text Available The chemiluminescence (CL signal immediately appeared when a hydrogen peroxide solution was injected into an iron-phthalocyanine tetrasulfonic acid (Fe-PTS aqueous solution. Moreover, the CL intensity of Fe-PTS decreased by adding L-tyrosine. Based on these results, the determination of trace amounts of L-tyrosine was developed using the quenching-chemiluminescence. The calibration curve of L-tyrosine was obtained in the concentration range of 2.0×10−7 M to 2.0×10−5 M. Moreover, the relative standard deviation (RSD was 1.63 % (=5 for 2.0×10−6 M L-tyrosine, and its detection limits (3σ were 1.81×10−7 M. The spike and recovery experiments for L-tyrosine were performed using a soft drink. Furthermore, the determination of L-tyrosine was applied to supplements containing various kinds of amino acids. Each satisfactory relative recovery was obtained at 98 to 102%.

  19. Acute phenylalanine/tyrosine depletion of phasic dopamine in the rat brain.

    Science.gov (United States)

    Shnitko, Tatiana A; Taylor, Sarah C; Stringfield, Sierra J; Zandy, Shannon L; Cofresí, Roberto U; Doherty, James M; Lynch, William B; Boettiger, Charlotte A; Gonzales, Rueben A; Robinson, Donita L

    2016-06-01

    Dopamine plays a critical role in striatal and cortical function, and depletion of the dopamine precursors phenylalanine and tyrosine is used in humans to temporarily reduce dopamine and probe the role of dopamine in behavior. This method has been shown to alter addiction-related behaviors and cognitive functioning presumably by reducing dopamine transmission, but it is unclear what specific aspects of dopamine transmission are altered. We performed this study to confirm that administration of an amino acid mixture omitting phenylalanine and tyrosine (Phe/Tyr[-]) reduces tyrosine tissue content in the prefrontal cortex (PFC) and nucleus accumbens (NAc), and to test the hypothesis that Phe/Tyr[-] administration reduces phasic dopamine release in the NAc. Rats were injected with a Phe/Tyr[-] amino acid mixture, a control amino acid mixture, or saline. High-performance liquid chromatography was used to determine the concentration of tyrosine, dopamine, or norepinephrine in tissue punches from the PFC and ventral striatum. In a separate group of rats, phasic dopamine release was measured with fast-scan cyclic voltammetry in the NAc core after injection with either the Phe/Tyr[-] mixture or the control amino acid solution. Phe/Tyr[-] reduced tyrosine content in the PFC and NAc, but dopamine and norepinephrine tissue content were not reduced. Moreover, Phe/Tyr[-] decreased the frequency of dopamine transients, but not their amplitude, in freely moving rats. These results indicate that depletion of tyrosine via Phe/Tyr[-] decreases phasic dopamine transmission, providing insight into the mechanism by which this method modifies dopamine-dependent behaviors in human imaging studies.

  20. Novel Tyrosine Phosphorylation Sites in Rat Skeletal Muscle Revealed by Phosphopeptide Enrichment and HPLC-ESI-MS/MS

    Science.gov (United States)

    Zhang, Xiangmin; Højlund, Kurt; Luo, Moulun; Meyer, Christian; Thangiah, Geetha; Yi, Zhengping

    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 (tyrosine phosphorylation sites have been identified in mammalian skeletal muscle to date. Here, we used immunoprecipitation of phosphotyrosine peptides prior to HPLC-ESI-MS/MS analysis to improve the discovery of tyrosine phosphorylation in relatively small skeletal muscle biopsies from rats. This resulted in the identification of 87 distinctly localized tyrosine phosphorylation sites in 46 muscle proteins. Among them, 31 appear to be novel. The tyrosine phosphorylated proteins included major enzymes in the glycolytic pathway and glycogen metabolism, sarcomeric proteins, and proteins involved in Ca2+ homeostasis and phosphocreatine resynthesis. Among proteins regulated by insulin, we found tyrosine phosphorylation sites in glycogen synthase, and two of its inhibitors, GSK-3α and DYRK1A. Moreover, tyrosine phosphorylation sites were identified in several MAP kinases and a protein tyrosine phosphatase, SHPTP2. These results provide the largest catalogue of mammalian skeletal muscle tyrosine phosphorylation sites to date and provide novel targets for the investigation of human skeletal muscle phosphoproteins in various disease states. PMID:22609512

  1. Microsomal aryl hydrocarbon hydroxylase comparison of the direct, indirect and radiometric assays

    International Nuclear Information System (INIS)

    Denison, M.S.; Murray, M.; Wilkinson, C.F.

    1983-01-01

    The direct fluorometric assay of aryl hydrocarbon hydroxlyase has been compared to the more commonly used indirect fluorometric and radiometric assays. Although rat hepatic microsomal activities measured by the direct assay were consistently higher than those obtained by the other assays, the relative changes in activity following enzyme induction and/or inhibition were similar. The direct assay provides an accurate and rapid measure of aryl hydrocarbon hydroxylase activity and avoids several problems inherent in the indirect and radiometric assays. 2 tables

  2. 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.

  3. 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...

  4. Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III Complexes

    Directory of Open Access Journals (Sweden)

    Jun Sumaoka

    2016-01-01

    Full Text Available Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear TbIII complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr, have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the TbIII ion as the emission center. Even in the coexistence of phosphoserine (pSer and phosphothreonine (pThr, pTyr can be efficintly detected with high selectivity. Simply by adding these TbIII complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates.

  5. Tyrosine Phosphorylation of Rac1: A Role in Regulation of Cell Spreading

    Science.gov (United States)

    Chang, Fumin; Lemmon, Christopher; Lietha, Daniel; Eck, Michael; Romer, Lewis

    2011-01-01

    Rac1 influences a multiplicity of vital cellular- and tissue-level control functions, making it an important candidate for targeted therapeutics. The activity of the Rho family member Cdc42 has been shown to be modulated by tyrosine phosphorylation at position 64. We therefore investigated consequences of the point mutations Y64F and Y64D in Rac1. Both mutations altered cell spreading from baseline in the settings of wild type, constitutively active, or dominant negative Rac1 expression, and were accompanied by differences in Rac1 targeting to focal adhesions. Rac1-Y64F displayed increased GTP-binding, increased association with βPIX, and reduced binding with RhoGDI as compared with wild type Rac1. Rac1-Y64D had less binding to PAK than Rac1-WT or Rac1-64F. In vitro assays demonstrated that Y64 in Rac1 is a target for FAK and Src. Taken together, these data suggest a mechanism for the regulation of Rac1 activity by non-receptor tyrosine kinases, with consequences for membrane extension. PMID:22163037

  6. 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

  7. Tyrosine Phosphorylation of the Guanine Nucleotide Exchange Factor GIV Promotes Activation of PI3K During Cell Migration

    Science.gov (United States)

    Lin, Changsheng; Ear, Jason; Pavlova, Yelena; Mittal, Yash; Kufareva, Irina; Ghassemian, Majid; Abagyan, Ruben; Garcia-Marcos, Mikel; Ghosh, Pradipta

    2014-01-01

    GIV (Gα-interacting vesicle-associated protein; also known as Girdin), enhances Akt activation downstream of multiple growth factor– and G-protein–coupled receptors to trigger cell migration and cancer invasion. Here we demonstrate that GIV is a tyrosine phosphoprotein that directly binds to and activates phosphoinositide 3-kinase (PI3K). Upon ligand stimulation of various receptors, GIV was phosphorylated at Tyr1764 and Tyr1798 by both receptor and non-receptor tyrosine kinases. These phosphorylation events enabled direct binding of GIV to the N- and C-terminal SH2 domains of p85α, a regulatory subunit of PI3K, stabilized receptor association with PI3K, and enhanced PI3K activity at the plasma membrane to trigger cell migration. Tyrosine phosphorylation of GIV and its association with p85α increased during metastatic progression of a breast carcinoma. These results suggest a mechanism by which multiple receptors activate PI3K through tyrosine phosphorylation of GIV, thereby making the GIVPI3K interaction a potential therapeutic target within the PI3K-Akt pathway. PMID:21954290

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

    Science.gov (United States)

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

    2017-05-01

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

  9. Tyrosine 842 in the activation loop is required for full transformation by the oncogenic mutant FLT3-ITD.

    Science.gov (United States)

    Kazi, Julhash U; Chougule, Rohit A; Li, Tianfeng; Su, Xianwei; Moharram, Sausan A; Rupar, Kaja; Marhäll, Alissa; Gazi, Mohiuddin; Sun, Jianmin; Zhao, Hui; Rönnstrand, Lars

    2017-07-01

    The type III receptor tyrosine kinase FLT3 is frequently mutated in acute myeloid leukemia. Oncogenic FLT3 mutants display constitutive activity leading to aberrant cell proliferation and survival. Phosphorylation on several critical tyrosine residues is known to be essential for FLT3 signaling. Among these tyrosine residues, Y842 is located in the so-called activation loop. The position of this tyrosine residue is well conserved in all receptor tyrosine kinases. It has been reported that phosphorylation of the activation loop tyrosine is critical for catalytic activity for some but not all receptor tyrosine kinases. The role of Y842 residue in FLT3 signaling has not yet been studied. In this report, we show that Y842 is not important for FLT3 activation or ubiquitination but plays a critical role in regulating signaling downstream of the receptor as well as controlling receptor stability. We found that mutation of Y842 in the FLT3-ITD oncogenic mutant background reduced cell viability and increased apoptosis. Furthermore, the introduction of the Y842 mutation in the FLT3-ITD background led to a dramatic reduction in in vitro colony forming capacity. Additionally, mice injected with cells expressing FLT3-ITD/Y842F displayed a significant delay in tumor formation, compared to FLT3-ITD expressing cells. Microarray analysis comparing gene expression regulated by FLT3-ITD versus FLT3-ITD/Y842F demonstrated that mutation of Y842 causes suppression of anti-apoptotic genes. Furthermore, we showed that cells expressing FLT3-ITD/Y842F display impaired activity of the RAS/ERK pathway due to reduced interaction between FLT3 and SHP2 leading to reduced SHP2 activation. Thus, we suggest that Y842 is critical for FLT3-mediated RAS/ERK signaling and cellular transformation.

  10. Src protein-tyrosine kinase structure and regulation

    International Nuclear Information System (INIS)

    Roskoski, Robert

    2004-01-01

    Src and Src-family protein kinases are proto-oncogenes that play key roles in cell morphology, motility, proliferation, and survival. v-Src (a viral protein) is encoded by the chicken oncogene of Rous sarcoma virus, and Src (the cellular homologue) is encoded by a physiological gene, the first of the proto-oncogenes. From the N- to C-terminus, Src contains an N-terminal 14-carbon myristoyl group, a unique segment, an SH3 domain, an SH2 domain, a protein-tyrosine kinase domain, and a C-terminal regulatory tail. The chief phosphorylation sites of Src include tyrosine 416 that results in activation from autophosphorylation and tyrosine 527 that results in inhibition from phosphorylation by C-terminal Src kinase. In the restrained state, the SH2 domain forms a salt bridge with phosphotyrosine 527, and the SH3 domain binds to the kinase domain via a polyproline type II left-handed helix. The SH2 and SH3 domains occur on the backside of the kinase domain away from the active site where they stabilize a dormant enzyme conformation. Protein-tyrosine phosphatases such as PTPα displace phosphotyrosine 527 from the Src SH2 domain and mediate its dephosphorylation leading to Src kinase activation. C-terminal Src kinase consists of an SH3, SH2, and kinase domain; it lacks an N-terminal myristoyl group and a C-terminal regulatory tail. Its X-ray structure has been determined, and the SH2 lobe occupies a position that is entirely different from that of Src. Unlike Src, the C-terminal Src kinase SH2 and SH3 domains stabilize an active enzyme conformation. Amino acid residues in the αD helix near the catalytic loop in the large lobe of C-terminal Src kinase serve as a docking site for the physiological substrate (Src) but not for an artificial substrate (polyGlu 4 Tyr)

  11. Photolysis mechanism of aqueous tyrosine upon excitation of the second absorption band

    International Nuclear Information System (INIS)

    Shimizu, O.

    1984-01-01

    The formation mechanism of tyrosinyl radical was studied for aqueous solutions of tyrosine under irradiation at 235 nm which falls into the second absorption band. The work is based upon the analysis of the rate of bityrosine production for steady-state excitation at low intensity. The results indicate that monophotonic O-H bond cleavage of tyrosine, presumably involving the upper excited triplet state, is the initial photoprocess leading to the tyrosinyl radical when tyrosine is excited into the second absorption band. (author)

  12. FAK tyrosine phosphorylation is regulated by AMPK and controls metabolism in human skeletal muscle

    DEFF Research Database (Denmark)

    Lassiter, David G; Nylén, Carolina; Sjögren, Rasmus J O

    2018-01-01

    the FAK gene, PTK2. RESULTS: AMPK activation reduced tyrosine phosphorylation of FAK in skeletal muscle. AICAR reduced p-FAKY397in isolated human skeletal muscle and cultured myotubes. Insulin stimulation did not alter FAK phosphorylation. Serum starvation increased AMPK activation, as demonstrated...

  13. C-Terminal Tyrosine Residue Modifications Modulate the Protective Phosphorylation of Serine 129 of α-Synuclein in a Yeast Model of Parkinson's Disease.

    Science.gov (United States)

    Kleinknecht, Alexandra; Popova, Blagovesta; Lázaro, Diana F; Pinho, Raquel; Valerius, Oliver; Outeiro, Tiago F; Braus, Gerhard H

    2016-06-01

    Parkinson´s disease (PD) is characterized by the presence of proteinaceous inclusions called Lewy bodies that are mainly composed of α-synuclein (αSyn). Elevated levels of oxidative or nitrative stresses have been implicated in αSyn related toxicity. Phosphorylation of αSyn on serine 129 (S129) modulates autophagic clearance of inclusions and is prominently found in Lewy bodies. The neighboring tyrosine residues Y125, Y133 and Y136 are phosphorylation and nitration sites. Using a yeast model of PD, we found that Y133 is required for protective S129 phosphorylation and for S129-independent proteasome clearance. αSyn can be nitrated and form stable covalent dimers originating from covalent crosslinking of two tyrosine residues. Nitrated tyrosine residues, but not di-tyrosine-crosslinked dimers, contributed to αSyn cytotoxicity and aggregation. Analysis of tyrosine residues involved in nitration and crosslinking revealed that the C-terminus, rather than the N-terminus of αSyn, is modified by nitration and di-tyrosine formation. The nitration level of wild-type αSyn was higher compared to that of A30P mutant that is non-toxic in yeast. A30P formed more dimers than wild-type αSyn, suggesting that dimer formation represents a cellular detoxification pathway in yeast. Deletion of the yeast flavohemoglobin gene YHB1 resulted in an increase of cellular nitrative stress and cytotoxicity leading to enhanced aggregation of A30P αSyn. Yhb1 protected yeast from A30P-induced mitochondrial fragmentation and peroxynitrite-induced nitrative stress. Strikingly, overexpression of neuroglobin, the human homolog of YHB1, protected against αSyn inclusion formation in mammalian cells. In total, our data suggest that C-terminal Y133 plays a major role in αSyn aggregate clearance by supporting the protective S129 phosphorylation for autophagy and by promoting proteasome clearance. C-terminal tyrosine nitration increases pathogenicity and can only be partially detoxified by

  14. Redox-Regulated Pathway of Tyrosine Phosphorylation Underlies NF-κB Induction by an Atypical Pathway Independent of the 26S Proteasome

    Science.gov (United States)

    Cullen, Sarah; Ponnappan, Subramaniam; Ponnappan, Usha

    2015-01-01

    Alternative redox stimuli such as pervanadate or hypoxia/reoxygenation, induce transcription factor NF-κB by phospho-tyrosine-dependent and proteasome-independent mechanisms. While considerable attention has been paid to the absence of proteasomal regulation of tyrosine phosphorylated IκBα, there is a paucity of information regarding proteasomal regulation of signaling events distinct from tyrosine phosphorylation of IκBα. To delineate roles for the ubiquitin-proteasome pathway in the phospho-tyrosine dependent mechanism of NF-κB induction, we employed the proteasome inhibitor, Aclacinomycin, and the phosphotyrosine phosphatase inhibitor, pervanadate (PV). Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported. Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB. Consistent with previous reports, a significant redox imbalance leading to the activation of tyrosine kinases, as occurs with pervanadate, is required for the induction of NF-κB. Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated. PMID:25671697

  15. Combinatorial analysis of enzymatic bottlenecks of L-tyrosine pathway by p-coumaric acid production in Saccharomyces cerevisiae.

    Science.gov (United States)

    Mao, Jiwei; Liu, Quanli; Song, Xiaofei; Wang, Hesuiyuan; Feng, Hui; Xu, Haijin; Qiao, Mingqiang

    2017-07-01

    To identify new enzymatic bottlenecks of L-tyrosine pathway for further improving the production of L-tyrosine and its derivatives. When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l -1 , respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l -1 ) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain. Combinatorial metabolic engineering provides a new strategy for further improvement of L-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.

  16. Induction by phenobarbital of aniline-p-hydroxylase in mouse liver under the influence of X-irradiation and 2,4,6-triethyleneimino-1,3,5-triazine

    International Nuclear Information System (INIS)

    Erger, M.; Hollatz, R.; Tempel, K.

    1977-01-01

    The phenobarbital-induced activity of aniline-p-hydroxylase in livers of mice was enhanced additionally when the animals were X-irradiated 4-16 hours before the administration of the inducer. The same effect could be demonstrated after repeated irradiation with low doses. 2,4,6-triethyleneimino-1,3,5-triazine (tretamine) inhibited the induction of aniline-p-hydroxylase only when administered in extremely high doses. Lower doses resulted in 'superinduciton'. (orig.) [de

  17. Modulation of catalytic activity in multi-domain protein tyrosine phosphatases.

    Directory of Open Access Journals (Sweden)

    Lalima L Madan

    Full Text Available Signaling mechanisms involving protein tyrosine phosphatases govern several cellular and developmental processes. These enzymes are regulated by several mechanisms which include variation in the catalytic turnover rate based on redox stimuli, subcellular localization or protein-protein interactions. In the case of Receptor Protein Tyrosine Phosphatases (RPTPs containing two PTP domains, phosphatase activity is localized in their membrane-proximal (D1 domains, while the membrane-distal (D2 domain is believed to play a modulatory role. Here we report our analysis of the influence of the D2 domain on the catalytic activity and substrate specificity of the D1 domain using two Drosophila melanogaster RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of Drosophila Leukocyte antigen Related (DLAR and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A. While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A reveal a conformational rationale for the experimental observations. These studies reveal that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain.

  18. A Novel Mutation in the CYP11B1 Gene Causes Steroid 11β-Hydroxylase Deficient Congenital Adrenal Hyperplasia with Reversible Cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Mohammad A. Alqahtani

    2015-01-01

    Full Text Available Congenital adrenal hyperplasia (CAH due to steroid 11β-hydroxylase deficiency is the second most common form of CAH, resulting from a mutation in the CYP11B1 gene. Steroid 11β-hydroxylase deficiency results in excessive mineralcorticoids and androgen production leading to hypertension, precocious puberty with acne, enlarged penis, and hyperpigmentation of scrotum of genetically male infants. In the present study, we reported 3 male cases from a Saudi family who presented with penile enlargement, progressive darkness of skin, hypertension, and cardiomyopathy. The elder patient died due to heart failure and his younger brothers were treated with hydrocortisone and antihypertensive medications. Six months following treatment, cardiomyopathy disappeared with normal blood pressure and improvement in the skin pigmentation. The underlying molecular defect was investigated by PCR-sequencing analysis of all coding exons and intron-exon boundary of the CYP11B1 gene. A novel biallelic mutation c.780 G>A in exon 4 of the CYP11B1 gene was found in the patients. The mutation created a premature stop codon at amino acid 260 (p.W260∗, resulting in a truncated protein devoid of 11β-hydroxylase activity. Interestingly, a somatic mutation at the same codon (c.779 G>A, p.W260∗ was reported in a patient with papillary thyroid cancer (COSMIC database. In conclusion, we have identified a novel nonsense mutation in the CYP11B1 gene that causes classic steroid 11β-hydroxylase deficient CAH. Cardiomyopathy and cardiac failure can be reversed by early diagnosis and treatment.

  19. Autophosphorylation of JAK2 on Tyrosines 221 and 570 Regulates Its Activity

    OpenAIRE

    Argetsinger, Lawrence S.; Kouadio, Jean-Louis K.; Steen, Hanno; Stensballe, Allan; Jensen, Ole N.; Carter-Su, Christin

    2004-01-01

    The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 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 a...

  20. Quantitative Tyrosine Phosphoproteomics of Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor-treated Lung Adenocarcinoma Cells Reveals Potential Novel Biomarkers of Therapeutic Response.

    Science.gov (United States)

    Zhang, Xu; Maity, Tapan; Kashyap, Manoj K; Bansal, Mukesh; Venugopalan, Abhilash; Singh, Sahib; Awasthi, Shivangi; Marimuthu, Arivusudar; Charles Jacob, Harrys Kishore; Belkina, Natalya; Pitts, Stephanie; Cultraro, Constance M; Gao, Shaojian; Kirkali, Guldal; Biswas, Romi; Chaerkady, Raghothama; Califano, Andrea; Pandey, Akhilesh; Guha, Udayan

    2017-05-01

    Mutations in the Epidermal growth factor receptor (EGFR) kinase domain, such as the L858R missense mutation and deletions spanning the conserved sequence 747 LREA 750 , are sensitive to tyrosine kinase inhibitors (TKIs). The gatekeeper site residue mutation, T790M accounts for around 60% of acquired resistance to EGFR TKIs. The first generation EGFR TKIs, erlotinib and gefitinib, and the second generation inhibitor, afatinib are FDA approved for initial treatment of EGFR mutated lung adenocarcinoma. The predominant biomarker of EGFR TKI responsiveness is the presence of EGFR TKI-sensitizing mutations. However, 30-40% of patients with EGFR mutations exhibit primary resistance to these TKIs, underscoring the unmet need of identifying additional biomarkers of treatment response. Here, we sought to characterize the dynamics of tyrosine phosphorylation upon EGFR TKI treatment of mutant EGFR-driven human lung adenocarcinoma cell lines with varying sensitivity to EGFR TKIs, erlotinib and afatinib. We employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry to identify and quantify tyrosine phosphorylated peptides. The proportion of tyrosine phosphorylated sites that had reduced phosphorylation upon erlotinib or afatinib treatment correlated with the degree of TKI-sensitivity. Afatinib, an irreversible EGFR TKI, more effectively inhibited tyrosine phosphorylation of a majority of the substrates. The phosphosites with phosphorylation SILAC ratios that correlated with the TKI-sensitivity of the cell lines include sites on kinases, such as EGFR-Y1197 and MAPK7-Y221, and adaptor proteins, such as SHC1-Y349/350, ERRFI1-Y394, GAB1-Y689, STAT5A-Y694, DLG3-Y705, and DAPP1-Y139, suggesting these are potential biomarkers of TKI sensitivity. DAPP1, is a novel target of mutant EGFR signaling and Y-139 is the major site of DAPP1 tyrosine phosphorylation. We also uncovered several off-target effects of these TKIs, such as MST1R-Y1238

  1. A role for the tyrosine kinase ACK1 in neurotrophin signaling and neuronal extension and branching

    Science.gov (United States)

    La Torre, A; del Mar Masdeu, M; Cotrufo, T; Moubarak, R S; del Río, J A; Comella, J X; Soriano, E; Ureña, J M

    2013-01-01

    Neurotrophins are involved in many crucial cellular functions, including neurite outgrowth, synapse formation, and plasticity. Although these events have long been known, the molecular determinants underlying neuritogenesis have not been fully characterized. Ack1 (activated Cdc42-associated tyrosine kinase) is a non-receptor tyrosine kinase that is highly expressed in the brain. Here, we demonstrate that Ack1 is a molecular constituent of neurotrophin signaling cascades in neurons and PC12 cells. We report that Ack1 interacts with Trk receptors and becomes tyrosine phosphorylated and its kinase activity is increased in response to neurotrophins. Moreover, our data indicate that Ack1 acts upstream of the Akt and MAPK pathways. We show that Ack1 overexpression induces neuritic outgrowth and promotes branching in neurotrophin-treated neuronal cells, whereas the expression of Ack1 dominant negatives or short-hairpin RNAs counteract neurotrophin-stimulated differentiation. Our results identify Ack1 as a novel regulator of neurotrophin-mediated events in primary neurons and in PC12 cells. PMID:23598414

  2. Complexes of γ-tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells

    International Nuclear Information System (INIS)

    Kukharskyy, Vitaliy; Sulimenko, Vadym; Macurek, Libor; Sulimenko, Tetyana; Draberova, Eduarda; Draber, Pavel

    2004-01-01

    Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that γ-tubulin (γ-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, γ-tubulin, and with anti-phosphotyrosine antibody revealed that γ-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in γ-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated γ-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing γ-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of γ-tubulin interaction with tubulin dimers or other proteins during neurogenesis

  3. Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase.

    Directory of Open Access Journals (Sweden)

    Saadat U Aleem

    Full Text Available The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution.

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

    Science.gov (United States)

    Mahajan, Monika; Yadav, Sudesh Kumar

    2014-08-01

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

  5. Evidence for a tyrosine protonation change during the primary phototransition of bacteriorhodopsin at low temperature.

    OpenAIRE

    Rothschild, K J; Roepe, P; Ahl, P L; Earnest, T N; Bogomolni, R A; Das Gupta, S K; Mulliken, C M; Herzfeld, J

    1986-01-01

    Isotopically labeled tyrosines have been selectively incorporated into bacteriorhodopsin (bR). A comparison of the low-temperature bR570 to K Fourier transform infrared-difference spectra of these samples and normal bR provides information about the role of tyrosine in the primary phototransition. Several tyrosine contributions to the difference spectrum are found. These results and comparison with the spectra of model compounds suggest that a tyrosinate group protonates during the bR570 to K...

  6. Combined quantum mechanical and molecular mechanical reaction pathway calculation for aromatic hydroxylation by p-hydroxybenzoate-3-hydroxylase

    NARCIS (Netherlands)

    Ridder, L.; Mulholland, A.; Rietjens, I.M.C.M.; Vervoort, J.

    1999-01-01

    The reaction pathway for the aromatic 3-hydroxylation of p-hydroxybenzoate by the reactive C4a-hydroperoxyflavin cofactor intermediate in p-hydroxybenzoate hydroxylase (PHBH) has been investigated by a combined quantum mechanical and molecular mechanical (QM/MM) method. A structural model for the

  7. Identification and analysis of a novel protein-tyrosine kinase from bovine thymus

    International Nuclear Information System (INIS)

    Zioncheck, T.F.; Harrison, M.L.; Geahlen, R.L.

    1986-01-01

    A cytosolic protein-tyrosine kinase has been identified and purified to near homogeneity from calf thymus by using the phosphorylation of the tyrosine-containing peptide angiotensin I as an assay. Specific peptide phosphorylating activity was enhanced by carrying out the assay at high ionic strength (2M NaCl). The inclusion of NaCl at this concentration acts to stimulate endogenous protein-tyrosine kinase activity while simultaneously inhibiting other endogenous kinases. The purification procedure involved extraction of the enzyme from calf-thymus and sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-sepharose, butylagarose, and Sephadex G-75. Analysis of the most highly purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single Coomassie blue-stained band of 41 KDa. This molecular weight was consistent with results obtained from gel filtration, indicating that the enzyme exists as a monomer. The enzyme has also been found to catalyze an autophosphorylation reaction. Incubation of the enzyme with Mn 2+ and [γ- 32 P]ATP led to its modification on a tyrosine residue. Phosphopeptide mapping experiments indicated that the 41 KDa kinase was distinct from p56, the major membrane-associated protein-tyrosine kinase in T lymphocytes

  8. A p130Cas tyrosine phosphorylated substrate domain decoy disrupts v-Crk signaling

    Directory of Open Access Journals (Sweden)

    Hanafusa Hidesaburo

    2002-07-01

    Full Text Available Abstract Background The adaptor protein p130Cas (Cas has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals. Results We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation. This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk. Conclusion Our approach suggests a novel method to study adaptor proteins that require phosphorylation, and indicates that mere tyrosine phosphorylation of the substrate domain of Cas is not sufficient for its function.

  9. Positron emission tomography imaging of adrenal masses: 18F-fluorodeoxyglucose and the 11β-hydroxylase tracer 11C-metomidate

    International Nuclear Information System (INIS)

    Zettinig, Georg; Becherer, Alexander; Pirich, Christian; Dudczak, Robert; Mitterhauser, Markus; Wadsak, Wolfgang; Kletter, Kurt; Vierhapper, Heinrich; Niederle, Bruno

    2004-01-01

    11 C-metomidate (MTO), a marker of 11β-hydroxylase, has been suggested as a novel positron emission tomography (PET) tracer for adrenocortical imaging. Up to now, experience with this very new tracer is limited. The aims of this study were (1) to evaluate this novel tracer, (2) to point out possible advantages in comparison with 18 F-fluorodeoxyglucose (FDG) and (3) to investigate in vivo the expression of 11β-hydroxylase in patients with primary aldosteronism. Sixteen patients with adrenal masses were investigated using both MTO and FDG PET imaging. All patients except one were operated on. Five patients had non-functioning adrenal masses, while 11 had functioning tumours(Cushing's syndrome, n=4; Conn's syndrome, n=5; phaeochromocytoma, n=2). Thirteen patients had benign disease, whereas in three cases the adrenal mass was malignant (adrenocortical cancer, n=1; malignant phaeochromocytoma, n=1; adrenal metastasis of renal cancer, n=1). MTO imaging clearly distinguished cortical from non-cortical adrenal masses (median standardised uptake values of 18.6 and 1.9, respectively, p<0.01). MTO uptake was slightly lower in patients with Cushing's syndrome than in those with Conn's syndrome, but the difference did not reach statistical significance. The expression of 11β-hydroxylase was not suppressed in the contralateral gland of patients with Conn's syndrome, whereas in Cushing's syndrome this was clearly the case. The single patient with adrenocortical carcinoma had MTO uptake in the lower range. MTO could not definitely distinguish between benign and malignant disease. FDG PET, however, identified clearly all three study patients with malignant adrenal lesions. We conclude: (1) MTO is an excellent imaging tool to distinguish adrenocortical and non-cortical lesions; (2) the in vivo expression of 11β-hydroxylase is lower in Cushing's syndrome than in Conn's syndrome, and there is no suppression of the contralateral gland in primary aldosteronism; (3) for the purpose

  10. Selective WGA uptake in the hippocampus from the locus coeruleus of DBH-WGA transgenic mice

    Directory of Open Access Journals (Sweden)

    Susan G eWalling

    2012-05-01

    Full Text Available We generated transgenic mice in which a transsynaptic tracer, wheat germ agglutinin (WGA, was specifically expressed in the locus coeruleus neurons under the control of the dopamine-β-hydroxylase gene promoter. WGA protein was produced in more than 95% of the tyrosine hydroxylase-positive locus coeruleus neurons sampled. Transynaptic transfer of WGA was most evident in CA3 neurons of the hippocampus, but appeared absent in CA1 neurons. Faint but significant WGA immunoreactivity was observed surrounding the nuclei of dentate granule cells. Putative hilar mossy cells, identified by the presence of calretinin in the ventral hippocampus, appeared uniformly positive for transynaptically transferred WGA protein. GAD67-positive interneurons in the hilar and CA3 regions tended to be WGA-positive, although a subset of them did not show WGA co-localization. The same mixed WGA uptake profile was apparent when examining co-localization with parvalbumin. The selective uptake of WGA by dentate granule cells, mossy cells and CA3 pyramidal neurons is consistent with evidence for a large proportion of conventional synapses adjacent to locus coeruleus axonal varicosities in these regions. The lack of WGA uptake in the CA1 region and its relatively sparse innervation by dopamine-β-hydroxylase-positive fibers suggest that a majority of the tyrosine hydroxylase-positive classical synapses revealed by electron microscopy in that region may be producing dopamine. The overall pattern of WGA uptake in these transgenic mice suggests a selective role for the granule cell-mossy cell-CA3 network in processing novelty or the salient environmental contingency changes signaled by locus coeruleus activity.

  11. Spectroscopic studies of fluorescent complexes of tyrosine 8-hydroxyquinoline and tyrosine-8-hydroxyquinaldine in aqueous phase

    International Nuclear Information System (INIS)

    Jakhrani, M.A.; Kazi, T.G.

    2002-01-01

    A new method has been developed by preparing complexes involving condensation of tyrosine with 8-hydroxyquinoline (Oxine) and 8-hydroxyquinaldine (Quinaldine) respectively, producing fluorescent products. The products obtained have been investigated for identification and quantitative estimation using different spectroscopic techniques including fluorescence activity of newly synthesized products. 8-hydroxyquinaldine and 8-hydroxyquinoline (Oxine) condensed with tyrosine separately produced water soluble fluorescent complexes. The complexes have been investigated for identification and quantitative estimation of amino acids. Identification of amino acids in nano mole or below than nano mole has become possible by present fluorometric activity of these complexes involving different excitation and emission wavelengths. The fluorometric activity of complexes has been observed to be 100 to 1000 times higher than assay method involving ninhydrin and amino acid analyzer. The method adopted in our laboratory is rapid, versatile with good reproducibility and provides excellent results for adoption by analytical, agricultural and biomedical laboratories to estimate amino acids and metals in composite matrix. (author)

  12. Tyrosine residues modification studied by MALDI-TOF mass spectrometry

    International Nuclear Information System (INIS)

    Santrucek, Jiri; Strohalm, Martin; Kadlcik, Vojtech; Hynek, Radovan; Kodicek, Milan

    2004-01-01

    Amino acid residue-specific reactivity in proteins is of great current interest in structural biology as it provides information about solvent accessibility and reactivity of the residue and, consequently, about protein structure and possible interactions. In the work presented tyrosine residues of three model proteins with known spatial structure are modified with two tyrosine-specific reagents: tetranitromethane and iodine. Modified proteins were specifically digested by proteases and the mass of resulting peptide fragments was determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Our results show that there are only small differences in the extent of tyrosine residues modification by tetranitromethane and iodine. However, data dealing with accessibility of reactive residues obtained by chemical modifications are not completely identical with those obtained by nuclear magnetic resonance and X-ray crystallography. These interesting discrepancies can be caused by local molecular dynamics and/or by specific chemical structure of the residues surrounding

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

    Directory of Open Access Journals (Sweden)

    Seeley TW

    2017-03-01

    Full Text Available Todd W Seeley, Mark D Sternlicht, Stephen J Klaus, Thomas B Neff, David Y Liu Therapeutics R&D, FibroGen, Inc., San Francisco, CA, USA Abstract: The effects of pharmacological hypoxia-inducible factor (HIF stabilization were investigated in the MMTV-Neundl-YD5 (NeuYD mouse model of breast cancer. This study first confirmed the sensitivity of this model to increased vascular endothelial growth factor (VEGF, using bigenic NeuYD;MMTV-VEGF-25 mice. Tumor initiation was dramatically accelerated in bigenic animals. Bigenic tumors were also more aggressive, with shortened doubling times and increased lung metastasis as compared to NeuYD controls. In separate studies, NeuYD mice were treated three times weekly from 7 weeks of age until study end with two different HIF prolyl hydroxylase inhibitors (HIF-PHIs, FG-4497 or roxadustat (FG-4592. In NeuYD mice, HIF-PHI treatments elevated erythropoiesis markers, but no differences were detected in tumor onset or the phenotypes of established tumors. Keywords: cancer progression, erythropoiesis, hypoxia-inducible factor, hypoxia-inducible factor prolyl hydroxylase inhibitors, vascular endothelial growth factor, MMTV-Neu breast cancer model

  14. 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

  15. 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.

  16. A quantum mechanical/molecular mechanical study of the hydroxylation of phenol and halogeneted derivatives by phenol hydroxylase

    NARCIS (Netherlands)

    Ridder, L.; Mulholland, A.J.; Rietjens, I.M.C.M.; Vervoort, J.

    2000-01-01

    A combined quantum mechanical and molecular mechanical (QM/MM) method (AM1/CHARMM) was used to investigate the mechanism of the aromatic hydroxylation of phenol by a flavin dependent phenol hydroxylase (PH), an essential reaction in the degradation of a wide range of aromatic compounds. The model

  17. PTP-PEST targets a novel tyrosine site in p120 catenin to control epithelial cell motility and Rho GTPase activity

    Science.gov (United States)

    Espejo, Rosario; Jeng, Yowjiun; Paulucci-Holthauzen, Adriana; Rengifo-Cam, William; Honkus, Krysta; Anastasiadis, Panos Z.; Sastry, Sarita K.

    2014-01-01

    ABSTRACT Tyrosine phosphorylation is implicated in regulating the adherens junction protein, p120 catenin (p120), however, the mechanisms are not well defined. Here, we show, using substrate trapping, that p120 is a direct target of the protein tyrosine phosphatase, PTP-PEST, in epithelial cells. Stable shRNA knockdown of PTP-PEST in colon carcinoma cells results in an increased cytosolic pool of p120 concomitant with its enhanced tyrosine phosphorylation and decreased association with E-cadherin. Consistent with this, PTP-PEST knockdown cells exhibit increased motility, enhanced Rac1 and decreased RhoA activity on a collagen substrate. Furthermore, p120 localization is enhanced at actin-rich protrusions and lamellipodia and has an increased association with the guanine nucleotide exchange factor, VAV2, and cortactin. Exchange factor activity of VAV2 is enhanced by PTP-PEST knockdown whereas overexpression of a VAV2 C-terminal domain or DH domain mutant blocks cell motility. Analysis of point mutations identified tyrosine 335 in the N-terminal domain of p120 as the site of PTP-PEST dephosphorylation. A Y335F mutant of p120 failed to induce the ‘p120 phenotype’, interact with VAV2, stimulate cell motility or activate Rac1. Together, these data suggest that PTP-PEST affects epithelial cell motility by controlling the distribution and phosphorylation of p120 and its availability to control Rho GTPase activity. PMID:24284071

  18. Metformin as a prevention and treatment for preeclampsia: effects on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion and endothelial dysfunction.

    Science.gov (United States)

    Brownfoot, Fiona C; Hastie, Roxanne; Hannan, Natalie J; Cannon, Ping; Tuohey, Laura; Parry, Laura J; Senadheera, Sevvandi; Illanes, Sebastian E; Kaitu'u-Lino, Tu'uhevaha J; Tong, Stephen

    2016-03-01

    explant-conditioned media (to whole vessels). Finally, we examined the effects of metformin on angiogenesis on maternal omental vessel explants. Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary endothelial cells, villous cytotrophoblast cells, and preterm preeclamptic placental villous explants. The reduction in soluble fms-like tyrosine kinase 1 and soluble endoglin secretion was rescued by coadministration of succinate, which suggests that the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin were likely to be regulated at the level of the mitochondria. In addition, the mitochondrial electron transport chain inhibitors rotenone and antimycin reduced soluble fms-like tyrosine kinase 1 secretion, which further suggests that soluble fms-like tyrosine kinase 1 secretion is regulated through the mitochondria. Mitochondrial electron transport chain activity in preterm preeclamptic placentas was increased compared with gestation-matched control subjects. Metformin improved features of endothelial dysfunction relevant to preeclampsia. It reduced endothelial cell messenger RNA expression of vascular cell adhesion molecule 1 that was induced by tumor necrosis factor-α (vascular cell adhesion molecule 1 is an inflammatory adhesion molecule up-regulated with endothelial dysfunction and is increased in preeclampsia). Placental conditioned media impaired bradykinin-induced vasodilation; this effect was reversed by metformin. Metformin also improved whole blood vessel angiogenesis impaired by fms-like tyrosine kinase 1. Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary human tissues, possibly by inhibiting the mitochondrial electron transport chain. The activity of the mitochondrial electron transport chain was increased in preterm preeclamptic placenta. Metformin reduced endothelial dysfunction, enhanced vasodilation in omental arteries, and induced

  19. The Dopamine D2 Receptor Gene in Lamprey, Its Expression in the Striatum and Cellular Effects of D2 Receptor Activation

    Science.gov (United States)

    Robertson, Brita; Huerta-Ocampo, Icnelia; Ericsson, Jesper; Stephenson-Jones, Marcus; Pérez-Fernández, Juan; Bolam, J. Paul; Diaz-Heijtz, Rochellys; Grillner, Sten

    2012-01-01

    All basal ganglia subnuclei have recently been identified in lampreys, the phylogenetically oldest group of vertebrates. Furthermore, the interconnectivity of these nuclei is similar to mammals and tyrosine hydroxylase-positive (dopaminergic) fibers have been detected within the input layer, the striatum. Striatal processing is critically dependent on the interplay with the dopamine system, and we explore here whether D2 receptors are expressed in the lamprey striatum and their potential role. We have identified a cDNA encoding the dopamine D2 receptor from the lamprey brain and the deduced protein sequence showed close phylogenetic relationship with other vertebrate D2 receptors, and an almost 100% identity within the transmembrane domains containing the amino acids essential for dopamine binding. There was a strong and distinct expression of D2 receptor mRNA in a subpopulation of striatal neurons, and in the same region tyrosine hydroxylase-immunoreactive synaptic terminals were identified at the ultrastructural level. The synaptic incidence of tyrosine hydroxylase-immunoreactive boutons was highest in a region ventrolateral to the compact layer of striatal neurons, a region where most striatal dendrites arborise. Application of a D2 receptor agonist modulates striatal neurons by causing a reduced spike discharge and a diminished post-inhibitory rebound. We conclude that the D2 receptor gene had already evolved in the earliest group of vertebrates, cyclostomes, when they diverged from the main vertebrate line of evolution (560 mya), and that it is expressed in striatum where it exerts similar cellular effects to that in other vertebrates. These results together with our previous published data (Stephenson-Jones et al. 2011, 2012) further emphasize the high degree of conservation of the basal ganglia, also with regard to the indirect loop, and its role as a basic mechanism for action selection in all vertebrates. PMID:22563388

  20. Mechanism of protein tyrosine phosphatase 1B-mediated inhibition of leptin signalling

    DEFF Research Database (Denmark)

    Lund, I K; Hansen, J A; Andersen, H S

    2005-01-01

    Upon leptin binding, the leptin receptor is activated, leading to stimulation of the JAK/STAT signal transduction cascade. The transient character of the tyrosine phosphorylation of JAK2 and STAT3 suggests the involvement of protein tyrosine phosphatases (PTPs) as negative regulators...

  1. Tyrosine Kinase Ligand-Receptor Pair Prediction by Using Support Vector Machine

    Directory of Open Access Journals (Sweden)

    Masayuki Yarimizu

    2015-01-01

    Full Text Available Receptor tyrosine kinases are essential proteins involved in cellular differentiation and proliferation in vivo and are heavily involved in allergic diseases, diabetes, and onset/proliferation of cancerous cells. Identifying the interacting partner of this protein, a growth factor ligand, will provide a deeper understanding of cellular proliferation/differentiation and other cell processes. In this study, we developed a method for predicting tyrosine kinase ligand-receptor pairs from their amino acid sequences. We collected tyrosine kinase ligand-receptor pairs from the Database of Interacting Proteins (DIP and UniProtKB, filtered them by removing sequence redundancy, and used them as a dataset for machine learning and assessment of predictive performance. Our prediction method is based on support vector machines (SVMs, and we evaluated several input features suitable for tyrosine kinase for machine learning and compared and analyzed the results. Using sequence pattern information and domain information extracted from sequences as input features, we obtained 0.996 of the area under the receiver operating characteristic curve. This accuracy is higher than that obtained from general protein-protein interaction pair predictions.

  2. Rat fetal ventral mesencephalon grown as solid tissue cultures

    DEFF Research Database (Denmark)

    Höglinger, G U; Sautter, J; Meyer, Morten

    1998-01-01

    in vitro (DIV) in the presence or absence (controls) of BDNF [100 ng/ml]. The dopamine content in the culture medium, analyzed by HPLC, was significantly higher (4-5 fold) in the BDNF group at DIV 8 and DIV 12 compared to the corresponding control levels (40 pg/ml). The number of tyrosine hydroxylase...

  3. 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.

  4. 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...... not require PTPase activity or posttranslational proteolytic cleavage of the R-PTP-kappa protein and is calcium independent. The results suggest that R-PTPases may provide a link between cell-cell contact and cellular signaling events involving tyrosine phosphorylation....

  5. SOCS proteins in regulation of receptor tyrosine kinase signaling

    DEFF Research Database (Denmark)

    Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar

    2014-01-01

    Receptor tyrosine kinases (RTKs) are a family of cell surface receptors that play critical roles in signal transduction from extracellular stimuli. Many in this family of kinases are overexpressed or mutated in human malignancies and thus became an attractive drug target for cancer treatment....... The signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar...

  6. Neurotoxicity of cerebro-spinal fluid from patients with Parkinson's disease on mesencephalic primary cultures as an in vitro model of dopaminergic neurons.

    Science.gov (United States)

    Kong, Ping; Zhang, Ben-Shu; Lei, Ping; Kong, Xiao-Dong; Zhang, Shi-Shuang; Li, Dai; Zhang, Yun

    2015-08-01

    Parkinson's disease is a degenerative disorder of the central nervous system. In spite of extensive research, neither the cause nor the mechanisms have been firmly established thus far. One assumption is that certain toxic substances may exist in the cerebro-spinal fluid (CSF) of Parkinson's disease patients. To confirm the neurotoxicity of CSF and study the potential correlation between neurotoxicity and the severity of Parkinson's disease, CSF was added to cultured cells. By observation of cell morphology, changes in the levels of lactate dehydrogenase, the ratio of tyrosine hydroxylase-positive cells, and the expression of tyrosine hydroxylase mRNA and protein, the differences between the two groups were shown. The created in vitro model of dopaminergic neurons using primary culture of mouse embryonic mesencephalic tissue is suitable for the study of neurotoxicity. The observations of the present study indicated that CSF from Parkinson's disease patients contains factors that can cause specific injury to cultured dopaminergic neurons. However, no obvious correlation was found between the neurotoxicity of CSF and the severity of Parkinson's disease.

  7. Ortho- and meta-tyrosine formation from phenylalanine in human saliva as a marker of hydroxyl radical generation during betel quid chewing.

    Science.gov (United States)

    Nair, U J; Nair, J; Friesen, M D; Bartsch, H; Ohshima, H

    1995-05-01

    The habit of betel quid chewing, common in South-East Asia and the South Pacific islands, is causally associated with an increased risk of oral cancer. Reactive oxygen species formed from polyphenolic betel quid ingredients and lime at alkaline pH have been implicated as the agents responsible for DNA and tissue damage. To determine whether hydroxyl radical (HO.) is generated in the human oral cavity during chewing of betel quid, the formation of o- and m-tyrosine from L-phenylalanine was measured. Both o- and m-tyrosine were formed in vitro in the presence of extracts of areca nut and/or catechu, transition metal ions such as Cu2+ and Fe2+ and lime or sodium carbonate (alkaline pH). Omission of any of these ingredients from the reaction mixture significantly reduced the yield of tyrosines. Hydroxyl radical scavengers such as ethanol, D-mannitol and dimethylsulfoxide inhibited the phenylalanine oxidation in a dose-dependent fashion. Five volunteers chewed betel quid consisting of betel leaf, areca nut, catechu and slaked lime (without tobacco). Their saliva, collected after chewing betel quid, contained high concentrations of p-tyrosine, but no appreciable amounts of o- or m-tyrosine. Saliva samples from the same subjects after chewing betel quid to which 20 mg phenylalanine had been added contained o- and m-tyrosine at concentrations ranging from 1010 to 3000 nM and from 1110 to 3140 nM respectively. These levels were significantly higher (P betel quid, which ranged from 14 to 70 nM for o-tyrosine and from 10 to 35 nM for m-tyrosine. These studies clearly demonstrate that the HO. radical is formed in the human oral cavity during betel quid chewing and is probably implicated in the genetic damage that has been observed in oral epithelial cells of chewers.

  8. Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Desai, D M; Sap, J; Schlessinger, J

    1993-01-01

    CD45, a transmembrane protein tyrosine phosphatase (PTPase), is required for TCR signaling. Multiple CD45 isoforms, differing in the extracellular domain, are expressed in a tissue- and activation-specific manner, suggesting an important function for this domain. We report that a chimeric protein...... that ligand-mediated regulation of receptor-PTPases may have mechanistic similarities with receptor tyrosine kinases....

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

    Energy Technology Data Exchange (ETDEWEB)

    Arnold, Frances H.

    2012-02-27

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

  10. Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 1. M412 and L550 intermediates

    International Nuclear Information System (INIS)

    Roepe, P.; Ahl, P.L.; Gupta, S.K.D.; Herzfeld, J.; Rothschild, K.J.

    1987-01-01

    The role of tyrosines in the bacteriorhodopsin (bR) photocycle has been investigated by using Fourier transform infrared (FTIR) and UV difference spectroscopies. Tyrosine contributions to the BR 570 → M 412 FTIR difference spectra recorded at several temperatures and pH's were identified by isotopically labeling tyrosine residues in bacteriorhodopsin. The frequencies and deuterium/hydrogen exchange sensitivities of these peaks and of peaks in spectra of model compounds in several environments suggest that at least two different tyrosine groups participate in the bR photocycle during the formation of M 412 . One group undergoes a tyrosinatetyrosine conversion during the BR 570 → K 630 transition. A second tyrosine group deprotonates between L 550 and M 412 . Low-temperature UV difference spectra in the 220-350-nm region of both purple membrane suspensions and rehydrated films support these conclusions. The UV spectra also indicate perturbations(s) of one or more tryptophan group(s). Several carboxyl groups appear to undergo a series of protonation changes between BR 570 and M 412 , as indicated by infrared absorption changes in the 1770-1720-cm -1 region. These results are consistent with the existence of a proton wire in bacteriorhodopsin that involves both tyrosine and carboxyl groups

  11. Development of amperometric L-tyrosine sensor based on Fe-doped hydroxyapatite nanoparticles

    International Nuclear Information System (INIS)

    Kanchana, P.; Lavanya, N.; Sekar, C.

    2014-01-01

    A novel biosensor based on Fe-doped hydroxyapatite (Fe-HA) nanoparticles and tyrosinase has been developed for the detection of L-tyrosine. Nanostructured Fe-HA was synthesized by a simple microwave irradiation method, and its phase formation, morphology and magnetic property were examined by powder X-ray diffraction (XRD), transmission electron microscopy (TEM) and vibrating sample magnetometer (VSM). Electrochemical performance of the nano Fe-HA/tyrosinase modified glassy carbon electrode (GCE) for detection of L-tyrosine was investigated by cyclic voltammetry (CV) and amperometric methods. The fabricated biosensor exhibited a linear response to L-tyrosine over a wide concentration range of 1.0 × 10 −7 to 1.0 × 10 −5 M with a detection limit of 245 nM at pH 7.0. In addition, the fabricated sensor showed an excellent selectivity, good reproducibility, long-term stability and anti-interference towards the determination of L-tyrosine. - Highlights: • A novel amperometric L-tyrosine biosensor has been fabricated using nanostructured Fe-HA. • The fabricated sensor exhibits a wide linear range, good stability and high reproducibility. • Fe-HA assists microenvironment and direct electron transfer between enzyme and electrode surface. • The nano Fe-HA and electrode fabrication procedure are simple and less expensive

  12. 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

  13. Unfolding of Ubiquitin Studied by Picosecond Time-Resolved Fluorescence of the Tyrosine Residue

    OpenAIRE

    Noronha, Melinda; Lima, João C.; Bastos, Margarida; Santos, Helena; Maçanita, António L.

    2004-01-01

    The photophysics of the single tyrosine in bovine ubiquitin (UBQ) was studied by picosecond time-resolved fluorescence spectroscopy, as a function of pH and along thermal and chemical unfolding, with the following results: First, at room temperature (25°C) and below pH 1.5, native UBQ shows single-exponential decays. From pH 2 to 7, triple-exponential decays were observed and the three decay times were attributed to the presence of tyrosine, a tyrosine-carboxylate hydrogen-bonded complex, and...

  14. PPAR/RXR Regulation of Fatty Acid Metabolism and Fatty Acid -Hydroxylase (CYP4 Isozymes: Implications for Prevention of Lipotoxicity in Fatty Liver Disease

    Directory of Open Access Journals (Sweden)

    James P. Hardwick

    2009-01-01

    Full Text Available Fatty liver disease is a common lipid metabolism disorder influenced by the combination of individual genetic makeup, drug exposure, and life-style choices that are frequently associated with metabolic syndrome, which encompasses obesity, dyslipidemia, hypertension, hypertriglyceridemia, and insulin resistant diabetes. Common to obesity related dyslipidemia is the excessive storage of hepatic fatty acids (steatosis, due to a decrease in mitochondria -oxidation with an increase in both peroxisomal -oxidation, and microsomal -oxidation of fatty acids through peroxisome proliferator activated receptors (PPARs. How steatosis increases PPAR activated gene expression of fatty acid transport proteins, peroxisomal and mitochondrial fatty acid -oxidation and -oxidation of fatty acids genes regardless of whether dietary fatty acids are polyunsaturated (PUFA, monounsaturated (MUFA, or saturated (SFA may be determined by the interplay of PPARs and HNF4 with the fatty acid transport proteins L-FABP and ACBP. In hepatic steatosis and steatohepatitis, the -oxidation cytochrome P450 CYP4A gene expression is increased even with reduced hepatic levels of PPAR. Although numerous studies have suggested the role ethanol-inducible CYP2E1 in contributing to increased oxidative stress, Cyp2e1-null mice still develop steatohepatitis with a dramatic increase in CYP4A gene expression. This strongly implies that CYP4A fatty acid -hydroxylase P450s may play an important role in the development of steatohepatitis. In this review and tutorial, we briefly describe how fatty acids are partitioned by fatty acid transport proteins to either anabolic or catabolic pathways regulated by PPARs, and we explore how medium-chain fatty acid (MCFA CYP4A and long-chain fatty acid (LCFA CYP4F -hydroxylase genes are regulated in fatty liver. We finally propose a hypothesis that increased CYP4A expression with a decrease in CYP4F genes may promote the progression of steatosis to

  15. Tyrosine kinase signalling in breast cancer

    International Nuclear Information System (INIS)

    Hynes, Nancy E

    2000-01-01

    Cells are continuously exposed to diverse stimuli ranging from soluble endocrine and paracrine factors to signalling molecules on neighbouring cells. Receptors of the tyrosine kinase family play an important role in the integration and interpretation of these external stimuli, allowing a cell to respond appropriately to its environment. The activation of receptor tyrosine kinases (RTKs) is tightly controlled, allowing a normal cell to correctly integrate its external environment with internal signal transduction pathways. In contrast, due to numerous molecular alterations arising during the course of malignancy, a tumour is characterized by an abnormal response to its environment, which allows cancer cells to evade the normal mechanisms controlling cellular proliferation. Alterations in the expression of various RTKs, in their activation, and in the signalling molecules lying downstream of the receptors play important roles in the development of cancer. This topic is the major focus of the thematic review section of this issue of Breast Cancer Research

  16. Evidence for association of the cloned liver growth hormone receptor with a tyrosine kinase

    DEFF Research Database (Denmark)

    Wang, X; Uhler, M D; Billestrup, N

    1992-01-01

    The ability of the cloned liver growth hormone (GH) receptor, when expressed in mammalian cell lines, to copurify with tyrosine kinase activity and be tyrosyl phosphorylated was examined. 125I-human growth hormone-GH receptor complexes isolated from COS-7 cells transiently expressing high levels...... of tyrosine kinase activity with cloned liver GH receptor. The level of phosphorylation of the GH receptor was very low, as compared with the endogenous GH receptor in 3T3-F442A cells, suggesting that tyrosine kinase activity is not intrinsic to the cloned GH receptor but rather resides with a kinase present...... in a variety of cell types. The finding that the level of phosphorylation of GH receptor appears to vary with cell type is consistent with the cloned liver GH receptor being a substrate for an associated tyrosine kinase and with the amount of such a GH receptor-associated tyrosine kinase being cell type-specific....

  17. Structural basis for the regulation mechanism of the tyrosine kinase CapB from Staphylococcus aureus.

    Directory of Open Access Journals (Sweden)

    Vanesa Olivares-Illana

    2008-06-01

    Full Text Available Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function.

  18. Therapeutic treatment with a novel hypoxia-inducible factor hydroxylase inhibitor (TRC160334 ameliorates murine colitis

    Directory of Open Access Journals (Sweden)

    Gupta R

    2014-01-01

    Full Text Available Ram Gupta,1 Anita R Chaudhary,2 Binita N Shah,1 Avinash V Jadhav,3 Shitalkumar P Zambad,1 Ramesh Chandra Gupta,4 Shailesh Deshpande,4 Vijay Chauthaiwale,4 Chaitanya Dutt4 1Department of Pharmacology, 2Cellular and Molecular Biology, 3Preclinical Safety Evaluation, 4Discovery, Torrent Research Centre, Torrent Pharmaceuticals Ltd, Gandhinagar, Gujarat, India Background and aim: Mucosal healing in inflammatory bowel disease (IBD can be achieved by improvement of intestinal barrier protection. Activation of hypoxia-inducible factor (HIF has been identified as a critical factor for barrier protection during mucosal insult and is linked with improvement in symptoms of colitis. Although prophylactic efficacy of HIF hydroxylase inhibitors in murine colitis have been established, its therapeutic efficacy in clinically relevant therapeutic settings have not been established. In the present study we aim to establish therapeutic efficacy of TRC160334, a novel HIF hydroxylase inhibitor, in animal models of colitis. Methods: The efficacy of TRC160334 was evaluated in two different mouse models of colitis by oral route. A prophylactic efficacy study was performed in a 2,4,6-trinitrobenzene sulfonic acid-induced mouse model of colitis representing human Crohn's disease pathology. Additionally, a therapeutic efficacy study was performed in a dextran sulfate sodium-induced mouse model of colitis, a model simulating human ulcerative colitis. Results: TRC160334 treatment resulted in significant improvement in disease end points in both models of colitis. TRC160334 treatment resulted into cytoprotective heatshock protein 70 induction in inflamed colon. TRC160334 successfully attenuated the rate of fall in body weight, disease activity index, and macroscopic and microscopic scores of colonic damage leading to overall improvement in study outcome. Conclusion: Our findings are the first to demonstrate that therapeutic intervention with a HIF hydroxylase inhibitor

  19. High dose sapropterin dihydrochloride therapy improves monoamine neurotransmitter turnover in murine phenylketonuria (PKU).

    Science.gov (United States)

    Winn, Shelley R; Scherer, Tanja; Thöny, Beat; Harding, Cary O

    2016-01-01

    Central nervous system (CNS) deficiencies of the monoamine neurotransmitters, dopamine and serotonin, have been implicated in the pathophysiology of neuropsychiatric dysfunction in phenylketonuria (PKU). Increased brain phenylalanine concentration likely competitively inhibits the activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), the rate limiting steps in dopamine and serotonin synthesis respectively. Tetrahydrobiopterin (BH4) is a required cofactor for TH and TPH activity. Our hypothesis was that treatment of hyperphenylalaninemic Pah(enu2/enu2) mice, a model of human PKU, with sapropterin dihydrochloride, a synthetic form of BH4, would stimulate TH and TPH activities leading to improved dopamine and serotonin synthesis despite persistently elevated brain phenylalanine. Sapropterin (20, 40, or 100mg/kg body weight in 1% ascorbic acid) was administered daily for 4 days by oral gavage to Pah(enu2/enu2) mice followed by measurement of brain biopterin, phenylalanine, tyrosine, tryptophan and monoamine neurotransmitter content. A significant increase in brain biopterin content was detected only in mice that had received the highest sapropterin dose, 100mg/kg. Blood and brain phenylalanine concentrations were unchanged by sapropterin therapy. Sapropterin therapy also did not alter the absolute amounts of dopamine and serotonin in brain but was associated with increased homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), dopamine and serotonin metabolites respectively, in both wild type and Pah(enu2/enu2) mice. Oral sapropterin therapy likely does not directly affect central nervous system monoamine synthesis in either wild type or hyperphenylalaninemic mice but may stimulate synaptic neurotransmitter release and subsequent metabolism. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Regulation of hematopoietic cell function by protein tyrosine kinase-encoding oncogenes, a review

    NARCIS (Netherlands)

    Punt, C. J.

    1992-01-01

    Tyrosine phosphorylation of proteins by protein tyrosine kinases (PTKs) is an important mechanism in the regulation of various cellular processes such as proliferation, differentiation, and transformation. Accumulating data implicate PTKs as essential intermediates in the transduction of

  1. Oxidation of protein tyrosine or methionine residues: From the amino acid to the peptide

    Energy Technology Data Exchange (ETDEWEB)

    Berges, J [Universite Pierre et Marie Curie, UMR 7616, Laboratoire de Chimie Theorique, 75005 Paris (France); Trouillas, P [EA 4021 Faculte de Pharmacie, 2 Rue du Dr. Marcland, 87025 Limoges Cedex (France); Houee-Levin, C, E-mail: jb@lct.jussieu.fr, E-mail: patrick.trouillas@unilim.fr, E-mail: chantal.houee@u-psud.fr [Universite Paris Sud, UMR 8000, Laboratoire de Chimie Physique, 91405 Orsay (France) (France)

    2011-01-01

    Methionine and tyrosine are competing targets of oxidizing free radicals in peptides or proteins. The first step is the addition of OH radicals either on the sulphur atom of methionine, followed by OH{sup -} elimination, or on the aromatic cycle of tyrosine. The next step can be stabilization of methionine radical cation by a two centre-three electron bond, or intramolecular electron transfer from tyrosine to the methionine radical cation. In this latter case a tyrosine radical is formed, which appears deprotonated. In a first step we have compared the stability of the OH radical adducts on Methionine or on Tyrosine. In agreement with experimental results, the thermodynamical data indicate that the OH adduct on Tyrosine and the radical cation are more stable than those on methionine. In a second step we have investigated the stabilization of the radical cations of Methionine by formation of intramolecular S:X two-center three-electron bond (X=S, N, O). Finally we have compared the spin densities on separated amino acids to that in a radical pentapeptide, methionine enkephalin. One observes a delocalisation of the orbital of the odd electron on the sulfur atom of Met and on the cycle of Tyr. The peptidic chain is also concerned.

  2. Oxidation of protein tyrosine or methionine residues: From the amino acid to the peptide

    International Nuclear Information System (INIS)

    Berges, J; Trouillas, P; Houee-Levin, C

    2011-01-01

    Methionine and tyrosine are competing targets of oxidizing free radicals in peptides or proteins. The first step is the addition of OH radicals either on the sulphur atom of methionine, followed by OH - elimination, or on the aromatic cycle of tyrosine. The next step can be stabilization of methionine radical cation by a two centre-three electron bond, or intramolecular electron transfer from tyrosine to the methionine radical cation. In this latter case a tyrosine radical is formed, which appears deprotonated. In a first step we have compared the stability of the OH radical adducts on Methionine or on Tyrosine. In agreement with experimental results, the thermodynamical data indicate that the OH adduct on Tyrosine and the radical cation are more stable than those on methionine. In a second step we have investigated the stabilization of the radical cations of Methionine by formation of intramolecular S:X two-center three-electron bond (X=S, N, O). Finally we have compared the spin densities on separated amino acids to that in a radical pentapeptide, methionine enkephalin. One observes a delocalisation of the orbital of the odd electron on the sulfur atom of Met and on the cycle of Tyr. The peptidic chain is also concerned.

  3. Tyrosine kinase fusion genes in pediatric BCR-ABL1-like acute lymphoblastic leukemia

    Science.gov (United States)

    Boer, Judith M.; Steeghs, Elisabeth M.P.; Marchante, João R.M.; Boeree, Aurélie; Beaudoin, James J.; Berna Beverloo, H.; Kuiper, Roland P.; Escherich, Gabriele; van der Velden, Vincent H.J.; van der Schoot, C. Ellen; de Groot-Kruseman, Hester A.; Pieters, Rob; den Boer, Monique L.

    2017-01-01

    Approximately 15% of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by gene expression similar to that of BCR-ABL1-positive disease and unfavorable prognosis. This BCR-ABL1-like subtype shows a high frequency of B-cell development gene aberrations and tyrosine kinase-activating lesions. To evaluate the clinical significance of tyrosine kinase gene fusions in children with BCP-ALL, we studied the frequency of recently identified tyrosine kinase fusions, associated genetic features, and prognosis in a representative Dutch/German cohort. We identified 14 tyrosine kinase fusions among 77 BCR-ABL1-like cases (18%) and none among 76 non-BCR-ABL1-like B-other cases. Novel exon fusions were identified for RCSD1-ABL2 and TERF2-JAK2. JAK2 mutation was mutually exclusive with tyrosine kinase fusions and only occurred in cases with high CRLF2 expression. The non/late response rate and levels of minimal residual disease in the fusion-positive BCR-ABL1-like group were higher than in the non-BCR-ABL1-like B-others (p<0.01), and also higher, albeit not statistically significant, compared with the fusion-negative BCR-ABL1-like group. The 8-year cumulative incidence of relapse in the fusion-positive BCR-ABL1-like group (35%) was comparable with that in the fusion-negative BCR-ABL1-like group (35%), and worse than in the non-BCR-ABL1-like B-other group (17%, p=0.07). IKZF1 deletions, predominantly other than the dominant-negative isoform and full deletion, co-occurred with tyrosine kinase fusions. This study shows that tyrosine kinase fusion-positive cases are a high-risk subtype of BCP-ALL, which warrants further studies with specific kinase inhibitors to improve outcome. PMID:27894077

  4. Ab initio study on electron excitation and electron transfer in tryptophan-tyrosine system

    International Nuclear Information System (INIS)

    Tong Jing; Li Xiangyuan

    2002-01-01

    In this article, ab initio calculation has been performed to evaluate the transition energy of electronic excitation in tryptophan and tyrosine by using semiempirical molecular orbital method AM1 and complete active space self-consistent field method. The solvent effect has been considered by means of the conductor-like screening model. After geometric optimizations of isolated tryptophan and tyrosine, and their corresponding radicals and cations, reaction heat of these electron transfer reactions have been obtained by the means of complete active space self-consistent field method. The transition energies from the ground state, respectively, to the lowest excited state and to the lowest triplet state of these two amino acids are also calculated and compared with the experimentally observed values. The ionization potential and electron affinity are also calculated for tryptophan and tyrosine employing Koopmans' theorem and ab initio calculation. Compared with the experimental measurements, the theoretical results are found satisfactory. Theoretical results give good explanations on the experimental phenomena that N 3 · can preferably oxide the side chain of tryptophan residue and then the electron transfer from tyrosine residue to tryptophan residue follows in peptides involving tryptophan and tyrosine

  5. Electrophoretic Detection and Confocal Microscopic Imaging of Tyrosine Nitrated Proteins in Plant Tissue.

    Science.gov (United States)

    Arora, Dhara; Singh, Neha; Bhatla, Satish C

    2018-01-01

    Tyrosine nitrated proteins can be detected in plant cells electrophoretically and their distribution can be monitored by confocal laser scanning microscopy (CLSM) imaging. One-dimensional polyacrylamide gel electrophoresis (1D PAGE) followed by Western blotting using polyclonal antibody against 3-nitrotyrosine residues enables detection of tyrosine nitrated proteins in plant cells. Here we describe detection of tyrosine nitrated proteins in the homogenates derived from sunflower (Helianthus annuus L.) seedling cotyledons. Total soluble proteins obtained from tissue homogenates are resolved using vertical gel electrophoresis followed by their electrophoretic transfer on to a microporous membrane support for immunodetection. Spatial distribution of tyrosine nitrated proteins can be visualized using an antibody against 3-nitrotyrosine residues. Immunofluorescent localization is performed by cutting 7 μm thick wax sections of tissue followed by incubation in primary anti-nitrotyrosine antibody (dilution 1:200) and secondary Cy-3 labeled anti-rabbit IgG antibody (dilution 1:1500). Confocal laser scanning microscopy analysis is undertaken using argon lasers (ex: 530-550 nm and em: 570 nm) at pinhole 1. Modulation in the abundance and spatial localization of tyrosine nitrated proteins in plant tissues can be monitored using these techniques.

  6. Adipocyte-specific protein tyrosine phosphatase 1B deletion increases lipogenesis, adipocyte cell size and is a minor regulator of glucose homeostasis.

    Directory of Open Access Journals (Sweden)

    Carl Owen

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B, a key negative regulator of leptin and insulin signaling, is positively correlated with adiposity and contributes to insulin resistance. Global PTP1B deletion improves diet-induced obesity and glucose homeostasis via enhanced leptin signaling in the brain and increased insulin signaling in liver and muscle. However, the role of PTP1B in adipocytes is unclear, with studies demonstrating beneficial, detrimental or no effect(s of adipose-PTP1B-deficiency on body mass and insulin resistance. To definitively establish the role of adipocyte-PTP1B in body mass regulation and glucose homeostasis, adipocyte-specific-PTP1B knockout mice (adip-crePTP1B(-/- were generated using the adiponectin-promoter to drive Cre-recombinase expression. Chow-fed adip-crePTP1B(-/- mice display enlarged adipocytes, despite having similar body weight/adiposity and glucose homeostasis compared to controls. High-fat diet (HFD-fed adip-crePTP1B(-/- mice display no differences in body weight/adiposity but exhibit larger adipocytes, increased circulating glucose and leptin levels, reduced leptin sensitivity and increased basal lipogenesis compared to controls. This is associated with decreased insulin receptor (IR and Akt/PKB phosphorylation, increased lipogenic gene expression and increased hypoxia-induced factor-1-alpha (Hif-1α expression. Adipocyte-specific PTP1B deletion does not beneficially manipulate signaling pathways regulating glucose homeostasis, lipid metabolism or adipokine secretion in adipocytes. Moreover, PTP1B does not appear to be the major negative regulator of the IR in adipocytes.

  7. Incorporation of Ortho- and Meta-Tyrosine Into Cellular Proteins Leads to Erythropoietin-Resistance in an Erythroid Cell Line

    Directory of Open Access Journals (Sweden)

    Esztella Mikolás

    2014-04-01

    Full Text Available Background/Aims: Erythropoietin-resistance is an unsolved concern in the treatment of renal anaemia. We aimed to investigate the possible role of ortho- and meta-tyrosine - the hydroxyl free radical products of L-phenylalanine - in the development of erythropoietin-resistance. Methods: TF-1 erythroblast cell line was used. Cell concentration was determined on day 1; 2 and 3 by two independent observers simultaneously in Bürker cell counting chambers. Protein concentration was determined with colorimetric method. Para-, ortho- and meta-tyrosine levels were measured using reverse phase-HPLC with fluorescence detection. Using Western blot method activating phosphorylation of STAT5 and ERK1/2 were investigated. Results: We found a time- and concentration-dependent decrease of erythropoietin-induced proliferative activity in case of ortho- and meta-tyrosine treated TF-1 erythroblasts, compared to the para-tyrosine cultured cells. Decreased erythropoietin-response could be regained with a competitive dose of para-tyrosine. Proteins of erythroblasts treated by ortho- or meta-tyrosine had lower para-tyrosine and higher ortho- or meta-tyrosine content. Activating phosphorylation of ERK and STAT5 due to erythropoietin was practically prevented by ortho- or meta-tyrosine treatment. Conclusion: According to this study elevated ortho- and meta-tyrosine content of erythroblasts may lead to the dysfunction of intracellular signaling, resulting in erythropoietin-hyporesponsiveness.

  8. Ethyl p-methoxycinnamate from Kaempferia galanga inhibits angiogenesis through tyrosine kinase

    Directory of Open Access Journals (Sweden)

    Juni Ekowati

    2015-04-01

    Full Text Available Background Many tumors express on their receptor tyrosine kinases vascular endothelial growth factor activity associated with angiogenesis. Inhibition of angiogenesis through reduction of tyrosine kinase activity is a promising strategy for cancer therapy. The present study aimed to determine the mechanism and potency of ethyl p-methoxycinnamate (EPMC isolated from Kaempferia galanga as angiogenesis inhibitor. Methods A laboratory experimental study was conducted using chorio-allantoic membranes (CAMs of nine-day old chicken eggs induced by 60ng basic fibroblast growth factor (bFGF. Ethyl p-methoxycinnamate (EPMC potency was determined at dosages of 30, 60, 90 and 120 mg and compared with celecoxib 60 mg as reference drug and one negative bFGF-induced control group. Neovascularization and endothelial cell count in CAM blood vessels were evaluated. To predict the antiangiogenic mechanism of EPMC, a docking study was performed with the Molegro Virtual Docker program on tyrosine kinase as receptor (PDB 1XKK. Results Angiogenesis stimulation by bFGF was prevented significantly (p<0.05 by EPMC at dosages of 30, 60, 90 and 120 mg and this activity was dose dependent. Molecular docking showed interaction between EPMC functional groups and tyrosine kinase amino acids at Met766, Met793, Thr854, Thr790, Gln791 and Ala743. There was an association between EPMC antiangiogenic activity and docking study results. Conclusions Ethyl p-methoxycinnamate is a potential new angiogenesis inhibitor through interaction with tyrosine kinase. EPMC could be a promising therapeutic agent for treatment of angiogenesis-related diseases.

  9. Protein-tyrosine phosphatase SHP2 contributes to GDNF neurotrophic activity through direct binding to phospho-Tyr687 in the RET receptor tyrosine kinase.

    Science.gov (United States)

    Perrinjaquet, Maurice; Vilar, Marçal; Ibáñez, Carlos F

    2010-10-08

    The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr(687) in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr(687) and association with components of the Tyr(1062) signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser(696), a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr(687) as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.

  10. 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

  11. Diversity of alkane hydroxylase genes on the rhizoplane of grasses planted in petroleum-contaminated soils

    OpenAIRE

    Tsuboi, Shun; Yamamura, Shigeki; Nakajima-Kambe, Toshiaki; Iwasaki, Kazuhiro

    2015-01-01

    The study investigated the diversity and genotypic features of alkane hydroxylase genes on rhizoplanes of grasses planted in artificial petroleum-contaminated soils to acquire new insights into the bacterial communities responsible for petroleum degradation in phytoremediation. Four types of grass (Cynodon dactylon, two phenotypes of Zoysia japonica, and Z. matrella) were used. The concentrations of total petroleum hydrocarbon effectively decreased in the grass-planted systems compared with t...

  12. Validation of o-tyrosine as a marker for detection and dosimetry of irradiated chicken meat

    International Nuclear Information System (INIS)

    Chuaqui-Offermanns, N.; McDougall, T.E.; Guerrero, A.M.

    1993-01-01

    The o-tyrosine has been proposed as a marker for postirradiation identification of food that contains protein. In this study, the validity of using o-tyrosine for this purpose has been tested and established. The validation process involved examination of background levels of o-tyrosine in unirradiated chicken, radiation dose yield, postirradiation storage, dose rate, radiation type, temperature during irradiation, and oxygen concentration during irradiation. The o-tyrosine is present in unirradiated chicken meat at variable levels. However, these background levels are low enough that o-tyrosine can serve to determine whether chicken has been irradiated or not at the commercially approved doses (3 kGy). The radiation dose response curve for the formation of o-tyrosine is linear. The apparent yields may vary with the analytical method used; however, it is independent of the dose rate, radiation type, atmosphere, and temperature (above freezing) during irradiation. It is also independent of the storage time and temperature after irradiation. It is concluded that this marker can be used to determine the absorbed dose in chicken meat irradiated with either gamma rays or electrons under normal or modified atmosphere

  13. PTB domain-directed substrate targeting in a tyrosine kinase from the unicellular choanoflagellate Monosiga brevicollis.

    Directory of Open Access Journals (Sweden)

    Victoria Prieto-Echagüe

    2011-04-01

    Full Text Available Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition.

  14. 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 (...

  15. Proton-Coupled Electron Transfer and a Tyrosine-Histidine Pair in a Photosystem II-Inspired β-Hairpin Maquette: Kinetics on the Picosecond Time Scale.

    Science.gov (United States)

    Pagba, Cynthia V; McCaslin, Tyler G; Chi, San-Hui; Perry, Joseph W; Barry, Bridgette A

    2016-02-25

    Photosystem II (PSII) and ribonucleotide reductase employ oxidation and reduction of the tyrosine aromatic ring in radical transport pathways. Tyrosine-based reactions involve either proton-coupled electron transfer (PCET) or electron transfer (ET) alone, depending on the pH and the pKa of tyrosine's phenolic oxygen. In PSII, a subset of the PCET reactions are mediated by a tyrosine-histidine redox-driven proton relay, YD-His189. Peptide A is a PSII-inspired β-hairpin, which contains a single tyrosine (Y5) and histidine (H14). Previous electrochemical characterization indicated that Peptide A conducts a net PCET reaction between Y5 and H14, which have a cross-strand π-π interaction. The kinetic impact of H14 has not yet been explored. Here, we address this question through time-resolved absorption spectroscopy and 280-nm photolysis, which generates a neutral tyrosyl radical. The formation and decay of the neutral tyrosyl radical at 410 nm were monitored in Peptide A and its variant, Peptide C, in which H14 is replaced by cyclohexylalanine (Cha14). Significantly, both electron transfer (ET, pL 11, L = lyonium) and PCET (pL 9) were accelerated in Peptide A and C, compared to model tyrosinate or tyrosine at the same pL. Increased electronic coupling, mediated by the peptide backbone, can account for this rate acceleration. Deuterium exchange gave no significant solvent isotope effect in the peptides. At pL 9, but not at pL 11, the reaction rate decreased when H14 was mutated to Cha14. This decrease in rate is attributed to an increase in reorganization energy in the Cha14 mutant. The Y5-H14 mechanism in Peptide A is reminiscent of proton- and electron-transfer events involving YD-H189 in PSII. These results document a mechanism by which proton donors and acceptors can regulate the rate of PCET reactions.

  16. Identification of a functional interaction between Kv4.3 channels and c-Src tyrosine kinase.

    Science.gov (United States)

    Gomes, Pedro; Saito, Tomoaki; Del Corsso, Cris; Alioua, Abderrahmane; Eghbali, Mansoureh; Toro, Ligia; Stefani, Enrico

    2008-10-01

    Voltage-gated K(+) (Kv) channels are key determinants of cardiac and neuronal excitability. A substantial body of evidence has accumulated in support of a role for Src family tyrosine kinases in the regulation of Kv channels. In this study, we examined the possibility that c-Src tyrosine kinase participates in the modulation of the transient voltage-dependent K(+) channel Kv4.3. Supporting a mechanistic link between Kv4.3 and c-Src, confocal microscopy analysis of HEK293 cells stably transfected with Kv4.3 showed high degree of co-localization of the two proteins at the plasma membrane. Our results further demonstrate an association between Kv4.3 and c-Src by co-immunoprecipitation and GST pull-down assays, this interaction being mediated by the SH2 and SH3 domains of c-Src. Furthermore, we show that Kv4.3 is tyrosine phosphorylated under basal conditions. The functional relevance of the observed interaction between Kv4.3 and c-Src was established in patch-clamp experiments, where application of the Src inhibitor PP2 caused a decrease in Kv4.3 peak current amplitude, but not the inactive structural analogue PP3. Conversely, intracellular application of recombinant c-Src kinase or the protein tyrosine phosphatase inhibitor bpV(phen) increased Kv4.3 peak current amplitude. In conclusion, our findings provide evidence that c-Src-induced Kv4.3 channel activation involves their association in a macromolecular complex and suggest a role for c-Src-Kv4.3 pathway in regulating cardiac and neuronal excitability.

  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. 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.

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

    Science.gov (United States)

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

    2013-03-18

    Non-classic adrenal hyperplasia (NCAH) has been associated with insulin resistance (IR). Therapies such as metformin, thiazolidinediones and lifestyle alterations improve IR and also ameliorate the biochemical and clinical abnormalities of NCAH, much as they do in polycystic ovarian syndrome (PCOS). More recently, bariatric surgery, such as Roux-en-Y gastric bypass (RYGBP), has also been associated with improvement in IR and amelioration of PCOS and may, therefore, be beneficial in NCAH. We report a case of a 39-year-old, deaf-mute, obese woman with NCAH due to 11-hydroxylase deficiency who underwent RYGBP followed by improvement of NCAH manifestations. She was initially treated with metformin and pioglitazone, which lowered serum 11-deoxycortisol from 198 ng/dl (irregular menses normalised as well. We conclude that RYGBP, like other interventions that reduce IR, may be another way of treating non-classic 11-hydroxylase deficiency in selected patients.

  20. Fluorescence anisotropy of tyrosinate anion using one-, two- and three-photon excitation: tyrosinate anion fluorescence.

    Science.gov (United States)

    Kierdaszuk, Borys

    2013-03-01

    We examined the emission spectra and steady-state anisotropy of tyrosinate anion fluorescence with one-photon (250-310 nm), two-photon (570-620 nm) and three-photon (750-930 nm) excitation. Similar emission spectra of the neutral (pH 7.2) and anionic (pH 13) forms of N-acetyl-L-tyrosinamide (NATyrA) (pKa 10.6) were observed for all modes of excitation, with the maxima at 302 and 352 nm, respectively. Two-photon excitation (2PE) and three-photon excitation (3PE) spectra of the anionic form were the same as that for one-photon excitation (1PE). In contrast, 2PE spectrum from the neutral form showed ~30-nm shift to shorter wavelengths relative to 1PE spectrum (λmax 275 nm) at two-photon energy (550 nm), the latter being overlapped with 3PE spectrum, both at two-photon energy (550 nm). Two-photon cross-sections for NATyrA anion at 565-580 nm were 10 % of that for N-acetyl-L-tryptophanamide (NATrpA), and increased to 90 % at 610 nm, while for the neutral form of NATyrA decreased from 2 % of that for NATrpA at 570 nm to near zero at 585 nm. Surprisingly, the fundamental anisotropy of NATyrA anion in vitrified solution at -60 °C was ~0.05 for 2PE at 610 nm as compared to near 0.3 for 1PE at 305 nm, and wavelength-dependence appears to be a basic feature of its anisotropy. In contrast, the 3PE anisotropy at 900 nm was about 0.5, and 3PE and 1PE anisotropy values appear to be related by the cos(6) θ to cos(2) θ photoselection factor (approx. 10/6) independently of excitation wavelength. Attention is drawn to the possible effect of tyrosinate anions in proteins on their multi-photon induced fluorescence emission and excitation spectra as well as excitation anisotropy spectra.