MOLECULAR CHARACTERIZATION OF BIOGENIC AMINE SYNTHESIS
Kent E Vrana, Elliot S. Vesell Professor And Chair
Wake Forest University Health Sciences Medical Center Blvd Winston-salem, Nc 27157
Grant 5R01GM038931-10 from National Institute Of General Medical Sciences IRG: NEUC
Abstract: Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting reactions in the biosynthesis of the catecholamines and serotonin, respectively. has formulated a working model of the subunit interactions responsible for TH and TPH tetramer formation in which sequence elements in both the N-terminal and C-terminal domains are required for creating mature tetrameric enzymes. Experiments are required to determine the specific residues involved in subunit assembly and enzyme activity as well as to determine the consequence of tetramer formation in a cellular environment. proposes experiments for both TH and TPH to test the hypothesis that higher order macromolecular structures are required for the allosteric regulation of enzyme activity (aim 1). Studies are proposed that will refine the current understanding of subunit assembly by employing more selective mutagenesis and that will focus on enzyme kinetics and phosphorylation-activation of the enzymes in vitro. In aim 2, the role of higher order enzyme structures in the cellular control of hydroxylase activity will be examined by transferring selected recombinant enzymes into eukaryotic cells. This approach is designed to assess the effects of quaternary structure (ie, monomer vs tetramer) on enzyme stability, as well as on regulation by endogenous second messenger pathways. In addition to providing valuable information concerning the basic biology of hydroxylase function, the principal investigator argues that these experiments will guide future efforts in transgenic animal production. In aim 3, proposes to extend his recent observations that the related hydroxylase, tyrosinase, acts as a quinone generator to convert dopamine into a covalent suicide inhibitor of TH. This observation not only suggests a new molecular basis for dopamine neurotoxicity, but provides a novel tool for identifying important active site amino acid residues. Utilizing radiolabeled dopamine, modified residues will be identified followed by the selective mutagenesis of these residues in recombinant TH. Given the highly homologous structures of TH and TPH, the findings of these studies with the former enzyme will then be extended to the latter. Given the nature of biogenic amines in human health, argues that it is important to understand the two primary biosynthetic enzymes. The proposed experiments are designed to extend s progress in this field and to enhance our knowledge of hydroxylase function.
Keywords: enzyme activity, recombinant protein, tryptophan 5 monooxygenase, tyrosine 3 monooxygenase, allosteric site, dopamine, posttranslational modification, quinone, PC12 cell, molecular cloning, site directed mutagenesis, tissue /cell culture
Project start date: 1991-01-01
Project end date: 2002-04-30
5R01GM038931-10 (2001): $161041
Sponsored Links Excellgen http://Excellgen.com
MOLECULAR CHARACTERIZATION OF TYROSINE HYDEOXYLASE
Kent E Vrana, Elliot S. Vesell Professor And Chair
Wake Forest University 1834 Wake Forest Road Winston-salem, Nc 27106
Grant 5R01GM038931-03 from National Institute Of General Medical Sciences IRG: NEUC
Abstract: Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. This pivotal enzyme is under strict regulatory control and responds to a variety of cellular stimuli by increasing its activity. In the short-term, TH is phosphorylated in vivo by at least three separate protein kinase systems (cAMP-dependent protein kinase, Ca++/phospholipid- dependent protein kinase and CA++/calmodulin-dependent protein kinase). TH can be phosphorylated in vitro by two additional kinases (cGMP-dependent protein kinase and TH-associated protein kinase from pheochromocytoma cells). Although the individual contributions of the separate kinases are unclear, the overall consequence of phosphorylation is an increase in enzyme activity. Five serine residues in rat pheochromocytoma TH have been identified as sites of phosphorylation. We propose to utilize a full-length cDNA clone (isolated in this laboratory) to test the functional significance of the phosphorylation of these serine residues. This cDNA clone has been expressed in bacteria as two different forms of beta-galactosidase fusion proteins. Both of these fusion enzymes exhibit high levels of activity. Attempts will be made to express TH as a native enzyme within bacteria. These recombinant enzymes will be purified and characterized as to their physical state, enzyme kinetics parameters and phosphorylation by protein kinases. Site specific mutagenesis will be employed to convert serine residues to a number of different amino acids. The altered enzymes will then be expressed and tested from their ability to be phosphorylated and activated by protein kinases. The role of each serine in TH activity and its post- translational regulation will be established. Deletion mutants will be constructed from each end of the TH cDNA. The prevailing hypothesis that TH is composed of an amino terminal regulatory domain and carboxyl terminal catalytic domain will be tested. This will also delineate the boundaries of the catalytic core of the enzyme. Both deletion and site specific point mutants will be constructed to test the hypothesis that leucine zippers are involved in the assemble of TH monomers into its native homotetramer form. We have identified two strong candidates for leucine zippers (with a third weaker candidate) in the carboxyl terminus of TH. These will be examined by interrupting the leucine repeat which is characteristic of the leucine zipper, expressing the enzyme and determining if it associates into multimers.
Keywords: enzyme activity, enzyme biosynthesis, phosphorylation, protein structure function, tyrosine 3 monooxygenase, calmodulin dependent protein kinase, chimeric protein, enzyme mechanism, enzyme structure, gene expression, leucine, neurotransmitter biosynthesis, protein kinase, protein kinase A, serine, laboratory rat, site directed mutagenesis, transfection
Project start date: 1991-01-01
Project end date: 1994-12-31
5R01GM038931-03 (1993): $99099
MOLECULAR CHARACTERIZATION OF BIOGENIC AMINE SYNTHESIS
Kent E Vrana, Elliot S. Vesell Professor And Chair
Wake Forest University 1834 Wake Forest Road Winston-salem, Nc 27106
Grant 5R01GM038931-06 from National Institute Of General Medical Sciences IRG: NEUC
Abstract: Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting reactions in the biosynthesis of the catecholamines and serotonin, respectively. These pivotal enzymes are under strict regulatory control and cells can respond to environmental stimuli either by modulating the activity of exiting enzyme molecules or by altering the number of enzyme molecules. TH possesses four serine residues which serve as phosphorylaiton substrates for sex distinct protein kinase systems. Although the phosphorylation of TPH is less well characterized, it is clear that this enzyme is subject to regulation by a least the cAMP-dependent and Ca+2/calmodulin-dependent protein kinases. Both TH and TPH exist in vivo as tetramers of identical subunits. However, little information exists on the structural forces underlying this subunit association. This laboratory has recently demonstrated that a carboxyl terminal leucine zipper is required for the assembly of TH dimers into tetramers. Although these leucine zipper repeats are conserved in TPH, their role in this enzyme remain unclear. These issues ill be pursued in three specific areas. For TH, (Aim #1) characterization of the role of leucine zippers in the structure/function of the enzyme will continue. Studies will concentrate on the involvement of tetrameric structure in the regulation of TH activity. Mutagenesis and bacterial expression of a rat cDNA clone will be employed to test the functions of two additional leucine repeats. For TPH, the recent expression of the rabbit enzyme in bacteria will be utilized to examine structure/function relationships. Specifically, (Aim #2) experiments will be conducted to test the hypothesis that TPH utilized a C-terminal leucine zipper for tetramer assembly. The roles of two additional leucine repeats will be assessed. Finally, (Aim #3) site- directed mutagenesis and traditional protein chemistry techniques will be brought to bear on the characterization of the phosphorylaiton regulation of TPH. In addition, chimera mutagenesis will be utilized to more fully examine the prevailing hypothesis that the enzyme consists of an N-terminal regulatory domain and C-terminal catalytic domain. Given the central nature of serotonin and the catecholamines in health and disease (e.g., hypertension and mental health), it is important to more fully characterize the enzymes which regulate their biosynthesis. The proposed experiments contribute to this understanding in areas for which there currently is limited information.
Keywords: enzyme complex, protein structure /function, tryptophan 5 monooxygenase, tyrosine 3 monooxygenase, allosteric site, gene deletion mutation, phosphoprotein, posttranslational modification, protein kinase, recombinant protein, chromatography, site directed mutagenesis, ultracentrifugation, western blotting
Project start date: 1991-01-01
Project end date: 1998-04-30
5R01GM038931-06 (1997): $121931
MOLECULAR CHARACTERIZATION OF BIOGENIC AMINE SYSTEMS
Kent E Vrana, Elliot S. Vesell Professor And Chair
Pennsylvania State Univ Hershey Med Ctr 500 University Drive Hershey, Pa 170330850
Grant 5R01GM038931-14 from National Institute Of General Medical Sciences IRG: ZRG1
Abstract: Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting reactions in the biosynthesis of the catecholamines and serotonin, respectively. During the previous funding cycle, work generated an innovative new structural model for TPH, provided insights into how the hydroxylases assemble into tetramers, identified a new phosphorylation site on TPH, and provided important information in the relative stabilities of TH and TPH. Experiments planned for the next funding period will address the need for more detailed information with a particular focus on four key areas. (1) Using the new structural model of TPH, along with available crystal coordinates for TH, sequence elements will be mapped within the active sites of the enzymes. These experiments will identify amino acid residues that influence and direct differential substrate utilization. (2) Mutagenesis will be conducted to refine the determinants of tetramer formation and the role of macromolecular assembly in regulating enzyme activity. Notably, while monomers have been demonstrated to retain activity, it remains to be determined how tetramer formation might influence the post-translational regulation of enzyme activity. (3) The hypothesis will be tested that differences in N-terminal regulatory domain sequences are responsible for enzyme stability. (4) The novel hypothesis will be explored that phosphorylation-regulation of TPH involves issues of selective stability as well as targeted interactions with the chaperone protein, 14-3-3. The proposed experiments extend the previous successes in the characterization of TH and TPH structure-function analysis. The studies will develop several novel ideas concerning the regulation of these enzymes and increase the base of knowledge from which we can interpret new information that will emanate from the human genome initiative in terms of naturally occurring polymorphisms in these important enzymes.
Keywords: genetic regulation, intermolecular interaction, model design /development, molecular assembly /self assembly, neurotransmitter biosynthesis, protein structure function, structural model, tryptophan 5 monooxygenase, tyrosine 3 monooxygenase, chemical kinetics, chemical stability, enzyme activity, enzyme mechanism, enzyme substrate, molecular site, phosphorylation, posttranslational modification, protein purification, site directed mutagenesis, tissue /cell culture
Project start date: 1991-01-01
Project end date: 2007-03-31
5R01GM038931-14 (2005): $187125
5R01GM038931-12 (2003): $180000
MOLECULAR CHARACTERIZATION OF BIOGENIC AMINE SYNTHESIS
Kent E Vrana, Elliot S. Vesell Professor And Chair
Wake Forest University Health Sciences Medical Center Blvd Winston-salem, Nc 27157
Grant 5R01GM038931-09 from National Institute Of General Medical Sciences IRG: NEUC
Abstract: Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting reactions in the biosynthesis of the catecholamines and serotonin, respectively. has formulated a working model of the subunit interactions responsible for TH and TPH tetramer formation in which sequence elements in both the N-terminal and C-terminal domains are required for creating mature tetrameric enzymes. Experiments are required to determine the specific residues involved in subunit assembly and enzyme activity as well as to determine the consequence of tetramer formation in a cellular environment. proposes experiments for both TH and TPH to test the hypothesis that higher order macromolecular structures are required for the allosteric regulation of enzyme activity (aim 1). Studies are proposed that will refine the current understanding of subunit assembly by employing more selective mutagenesis and that will focus on enzyme kinetics and phosphorylation-activation of the enzymes in vitro. In aim 2, the role of higher order enzyme structures in the cellular control of hydroxylase activity will be examined by transferring selected recombinant enzymes into eukaryotic cells. This approach is designed to assess the effects of quaternary structure (ie, monomer vs tetramer) on enzyme stability, as well as on regulation by endogenous second messenger pathways. In addition to providing valuable information concerning the basic biology of hydroxylase function, the principal investigator argues that these experiments will guide future efforts in transgenic animal production. In aim 3, proposes to extend his recent observations that the related hydroxylase, tyrosinase, acts as a quinone generator to convert dopamine into a covalent suicide inhibitor of TH. This observation not only suggests a new molecular basis for dopamine neurotoxicity, but provides a novel tool for identifying important active site amino acid residues. Utilizing radiolabeled dopamine, modified residues will be identified followed by the selective mutagenesis of these residues in recombinant TH. Given the highly homologous structures of TH and TPH, the findings of these studies with the former enzyme will then be extended to the latter. Given the nature of biogenic amines in human health, argues that it is important to understand the two primary biosynthetic enzymes. The proposed experiments are designed to extend s progress in this field and to enhance our knowledge of hydroxylase function.
Keywords: enzyme activity, recombinant protein, tryptophan 5 monooxygenase, tyrosine 3 monooxygenase, allosteric site, dopamine, posttranslational modification, quinone, PC12 cell, molecular cloning, site directed mutagenesis, tissue /cell culture
Project start date: 1991-01-01
Project end date: 2002-04-30
5R01GM038931-09 (2000): $156352
5R01GM038931-08 (1999): $151798
MOLECULAR CHARACTERIZATION OF HUMAN TRYPTOPHAN HYDROXYLASE 2 (HTPH2)
Kent E Vrana, Professor
Pennsylvania State Univ Hershey Med Ctr, 500 University Drive, Hershey, Pa 17033-0850
Grant 5R01GM038931-18 from National Institute Of General Medical Sciences
Abstract: Tryptophan hydroxylase (TPH) catalyzes the initial and rate-limiting step in the biosynthesis of serotonin. As such, it has been implicated in a variety of mental health disorders. One of the goals of this long-standing R01 has been to better understand this pivotal enzyme. However, in the midst of the preceding cycle, other investigators (and current collaborators) made the seminal discovery of a new variant of this enzyme (encoded by a separate gene) that is responsible for central nervous system serotonin synthesis - TPH2. Given that the work in this field had to this point focused on the peripheral enzyme (now termed TPH1), and given the importance of CNS serotonin to health and disease, we propose shifting the emphasis of this upcoming renewal period to better understanding this novel and pivotal enzyme. Virtually nothing is known about the structure, function, and enzymology of hTPH2. Partnering with the discoverers of this new enzyme, we have established important new characterization studies on the human TPH2 (hTPH2). We are strongly positioned to pursue three specific aims in the present proposal. Specific Aim 1 will characterize the functional consequences of naturally-occurring polymorphisms in the hTPH2 gene. In the few short years since discovery of the TPH2 gene, seven different coding region polymorphisms have been described. Little is known concerning the consequences of these amino acid substitutions and this aim will address this gap in our knowledge. Aim 2 will use data we have obtained from tyrosine hydroxylase and hTPH1 to map the active site of hTPH2. These biochemical studies will provide important functional insights into this novel and important enzyme. Specific Aim 3 will explore the regulation of hTPH2 by its N-terminal regulatory domain. These experiments will contribute to our knowledge of the dynamic regulation of serotonin biosynthesis in health and disease. The novel hTPH2 gene was first described three years ago. Its discovery resolves several important discprepancies in the field of serotonin biosynthese. However, we must establish a deeper understanding of this pivotal enzyme to better appreciate its role in health and disease and to provide a basis for potential development of novel pharmacotherapeutics
Keywords: 3-(2-Aminoethyl)-1H-indol-5-ol; 5-HT; 5-Hydroxytryptamine; 5HT; Active Sites; Address; Amino Acid Substitution; Amino Acids; Amino Acids, Aromatic; Anabolism; Area; Aromatic Amino Acids; Assay; Base Sequence; Bioassay; Biochemical; Biogenic Amines; Biologic Assays; Biological Assay; Biosynthetic Proteins; Catecholamines; Cell Culture Techniques; Central Nervous System; Code; Coding System; Crystallographies; Crystallography; Data; Depression; Development; Disease; Disorder; Enteramine; Enzymatic Biochemistry; Enzyme Stability; Enzymes; Enzymology; Functional RNA; Funding; Genes; Genetic Alteration; Genetic Change; Genetic Polymorphism; Genetic defect; Genetics-Mutagenesis; Goals; Grant; Health; Hippophaine; History; Human; Human, General; Hydroxylases; Image; Investigators; Kinetic; Kinetics; Knowledge; L-Phenylalanine; L-Tryptophan; L-Tyrosine; L-Tyrosine, tetrahydrobiopterin[{..}]oxygen oxidoreductase (3-hydroxylating); L-tryptophan, tetrahydropteridine[{..}]oxygen oxidoreductase (5-hydroxylating); Laboratories; Levotryptophan; Mammalian Cell; Man (Taxonomy); Man, Modern; Maps; Mental Depression; Mental Health; Mental Hygiene; Mental disorders; Mental health disorders; Mixed Function Oxidases; Mixed Function Oxygenases; Modeling; Models, Molecular; Molecular; Molecular Biology, Mutagenesis; Molecular Models; Monooxygenases; Mutagenesis; Mutation; N-terminal; NH2-terminal; NRVS-SYS; Nervous System; Nervous System, CNS; Nervous system structure; Neuraxis; Neurologic Body System; Neurologic Organ System; Non-Coding; Non-Coding RNA; Nucleic Acid Biochemistry, Molecular Modeling; Nucleotide Sequence; PC-12; PC12 Cells; Peripheral; Phenylalanine; Phenylalanine, L-Isomer; Pheochromocytoma Cell Line; Phosphorylation Site; Polymorphism (Genetics); Polymorphism, Genetic; Position; Positioning Attribute; Programs (PT); Programs [Publication Type]; Protein/Amino Acid Biochemistry, Molecular Modeling; Psychiatric Disease; Psychiatric Disorder; Psychological Health; Publishing; Recombinant Proteins; Recording of previous events; Regulation; Research Personnel; Researchers; Role; Seminal; Serotonin; Silent Mutation; Site; Sites, Active; Structure; Structure-Activity Relationship; Substance abuse problem; Sympathins; TDO; TDO2 gene; TPH2; TYR; Testing; Time; Tph2 protein, human; Tryptophan; Tryptophan 5-monooxygenase; Tryptophan Hydroxylase; Tryptophan Monooxygenase; Tyrosine; Tyrosine 3-Monooxygenase; Tyrosine Hydroxylase; Tyrosine, L-isomer; Unspecified Mental Disorder; Variant; Variation; Work; abuse of substances; aminoacid; base; biosynthesis; chemical structure function; design; designing; disease/disorder; enzyme activity; experiment; experimental research; experimental study; gene discovery; genome mutation; human tryptophan hydroxylase 2; imaging; inhibitor; inhibitor/antagonist; insight; mental illness; molecular modeling; mutant; novel; nucleic acid sequence; para-Tyrosine; pheochromocytoma 12 cell line; polymorphism; programs; psychological disorder; research study; social role; structure function relationship; substance abuse; tryptophan 5 hydroxylase; tryptophan hydroxylase 2, human
Project start date: 1991-01-01
Project end date: 2011-02-28
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
5R01GM038931-18 (2010): $187441
MOLECULAR CHARACTERIZATION OF BIOGENIC AMINE SYNTHESIS
Kent E Vrana, Elliot S. Vesell Professor And Chair
Wake Forest University 1834 Wake Forest Road Winston-salem, Nc 27106
Grant 5R01GM038931-05 from National Institute Of General Medical Sciences IRG: NEUC
Project start date: 1991-01-01
Project end date: 1998-03-31
5R01GM038931-05 (1996): $118175
Grants awarded to Kent E Vrana
MOLECULAR CHARACTERIZATION OF TYROSINE HYDEOXYLASE
Kent E Vrana, Elliot S. Vesell Professor And Chair
Wake Forest University 1834 Wake Forest Road Winston-salem, Nc 27106
Grant 7R01GM038931-02 from National Institute Of General Medical Sciences IRG: NEUC
Abstract: Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. This pivotal enzyme is under strict regulatory control and responds to a variety of cellular stimuli by increasing its activity. In the short-term, TH is phosphorylated in vivo by at least three separate protein kinase systems (cAMP-dependent protein kinase, Ca++/phospholipid- dependent protein kinase and CA++/calmodulin-dependent protein kinase). TH can be phosphorylated in vitro by two additional kinases (cGMP-dependent protein kinase and TH-associated protein kinase from pheochromocytoma cells). Although the individual contributions of the separate kinases are unclear, the overall consequence of phosphorylation is an increase in enzyme activity. Five serine residues in rat pheochromocytoma TH have been identified as sites of phosphorylation. We propose to utilize a full-length cDNA clone (isolated in this laboratory) to test the functional significance of the phosphorylation of these serine residues. This cDNA clone has been expressed in bacteria as two different forms of beta-galactosidase fusion proteins. Both of these fusion enzymes exhibit high levels of activity. Attempts will be made to express TH as a native enzyme within bacteria. These recombinant enzymes will be purified and characterized as to their physical state, enzyme kinetics parameters and phosphorylation by protein kinases. Site specific mutagenesis will be employed to convert serine residues to a number of different amino acids. The altered enzymes will then be expressed and tested from their ability to be phosphorylated and activated by protein kinases. The role of each serine in TH activity and its post- translational regulation will be established. Deletion mutants will be constructed from each end of the TH cDNA. The prevailing hypothesis that TH is composed of an amino terminal regulatory domain and carboxyl terminal catalytic domain will be tested. This will also delineate the boundaries of the catalytic core of the enzyme. Both deletion and site specific point mutants will be constructed to test the hypothesis that leucine zippers are involved in the assemble of TH monomers into its native homotetramer form. We have identified two strong candidates for leucine zippers (with a third weaker candidate) in the carboxyl terminus of TH. These will be examined by interrupting the leucine repeat which is characteristic of the leucine zipper, expressing the enzyme and determining if it associates into multimers.
Keywords: enzyme activity, enzyme biosynthesis, phosphorylation, protein structure function, tyrosine 3 monooxygenase, calmodulin dependent protein kinase, chimeric protein, cyclic AMP dependent protein kinase, enzyme mechanism, enzyme structure, gene expression, leucine, neurotransmitter biosynthesis, protein kinase, serine, laboratory rat, site directed mutagenesis, transfection
Project start date: 1991-01-01
Project end date: 1993-12-31
7R01GM038931-02 (1992): $97858
MOLECULAR CHARACTERIZATION OF BIOGENIC AMINE SYNTHESIS
Kent E Vrana, Elliot S. Vesell Professor And Chair
Wake Forest University 1834 Wake Forest Road Winston-salem, Nc 27106
Grant 2R01GM038931-04A2 from National Institute Of General Medical Sciences IRG: NEUC
Abstract: Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting reactions in the biosynthesis of the catecholamines and serotonin, respectively. These pivotal enzymes are under strict regulatory control and cells can respond to environmental stimuli either by modulating the activity of exiting enzyme molecules or by altering the number of enzyme molecules. TH possesses four serine residues which serve as phosphorylaiton substrates for sex distinct protein kinase systems. Although the phosphorylation of TPH is less well characterized, it is clear that this enzyme is subject to regulation by a least the cAMP-dependent and Ca+2/calmodulin-dependent protein kinases. Both TH and TPH exist in vivo as tetramers of identical subunits. However, little information exists on the structural forces underlying this subunit association. This laboratory has recently demonstrated that a carboxyl terminal leucine zipper is required for the assembly of TH dimers into tetramers. Although these leucine zipper repeats are conserved in TPH, their role in this enzyme remain unclear. These issues ill be pursued in three specific areas. For TH, (Aim #1) characterization of the role of leucine zippers in the structure/function of the enzyme will continue. Studies will concentrate on the involvement of tetrameric structure in the regulation of TH activity. Mutagenesis and bacterial expression of a rat cDNA clone will be employed to test the functions of two additional leucine repeats. For TPH, the recent expression of the rabbit enzyme in bacteria will be utilized to examine structure/function relationships. Specifically, (Aim #2) experiments will be conducted to test the hypothesis that TPH utilized a C-terminal leucine zipper for tetramer assembly. The roles of two additional leucine repeats will be assessed. Finally, (Aim #3) site- directed mutagenesis and traditional protein chemistry techniques will be brought to bear on the characterization of the phosphorylaiton regulation of TPH. In addition, chimera mutagenesis will be utilized to more fully examine the prevailing hypothesis that the enzyme consists of an N-terminal regulatory domain and C-terminal catalytic domain. Given the central nature of serotonin and the catecholamines in health and disease (e.g., hypertension and mental health), it is important to more fully characterize the enzymes which regulate their biosynthesis. The proposed experiments contribute to this understanding in areas for which there currently is limited information.
Keywords: enzyme complex, protein structure function, tryptophan 5 monooxygenase, tyrosine 3 monooxygenase, allosteric site, gene deletion mutation, phosphoprotein, posttranslational modification, protein kinase, recombinant protein, chromatography, site directed mutagenesis, ultracentrifugation, western blotting
Project start date: 1991-01-01
Project end date: 1998-03-31
2R01GM038931-04A2 (1995): $117125
MOLECULAR CHARACTERIZATION OF BIOGENIC AMINE SYSTEMS
Kent E Vrana, Elliot S. Vesell Professor And Chair
Wake Forest University Health Sciences Medical Center Blvd Winston-salem, Nc 27157
Grant 2R01GM038931-11 from National Institute Of General Medical Sciences IRG: ZRG1
Abstract: Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting reactions in the biosynthesis of the catecholamines and serotonin, respectively. During the previous funding cycle, work generated an innovative new structural model for TPH, provided insights into how the hydroxylases assemble into tetramers, identified a new phosphorylation site on TPH, and provided important information in the relative stabilities of TH and TPH. Experiments planned for the next funding period will address the need for more detailed information with a particular focus on four key areas. (1) Using the new structural model of TPH, along with available crystal coordinates for TH, sequence elements will be mapped within the active sites of the enzymes. These experiments will identify amino acid residues that influence and direct differential substrate utilization. (2) Mutagenesis will be conducted to refine the determinants of tetramer formation and the role of macromolecular assembly in regulating enzyme activity. Notably, while monomers have been demonstrated to retain activity, it remains to be determined how tetramer formation might influence the post-translational regulation of enzyme activity. (3) The hypothesis will be tested that differences in N-terminal regulatory domain sequences are responsible for enzyme stability. (4) The novel hypothesis will be explored that phosphorylation-regulation of TPH involves issues of selective stability as well as targeted interactions with the chaperone protein, 14-3-3. The proposed experiments extend the previous successes in the characterization of TH and TPH structure-function analysis. The studies will develop several novel ideas concerning the regulation of these enzymes and increase the base of knowledge from which we can interpret new information that will emanate from the human genome initiative in terms of naturally occurring polymorphisms in these important enzymes.
Keywords: genetic regulation, intermolecular interaction, model design /development, molecular assembly /self assembly, neurotransmitter biosynthesis, protein structure function, structural model, tryptophan 5 monooxygenase, tyrosine 3 monooxygenase, chemical kinetics, chemical stability, enzyme activity, enzyme mechanism, enzyme substrate, molecular site, phosphorylation, posttranslational modification, protein purification, site directed mutagenesis, tissue /cell culture
Project start date: 1991-01-01
Project end date: 2006-04-30
2R01GM038931-11 (2002): $180156
3R01GM038931-14S1 (2006): $61042
7R01GM038931-13 (2004): $179531
MOLECULAR CHARACTERIZATION OF BIOGENIC AMINE SYNTHESIS
Kent E Vrana, Elliot S. Vesell Professor And Chair
Wake Forest University 1834 Wake Forest Road Winston-salem, Nc 27106
Grant 2R01GM038931-07 from National Institute Of General Medical Sciences IRG: NEUC
Abstract: Tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) catalyze the rate-limiting reactions in the biosynthesis of the catecholamines and serotonin, respectively. has formulated a working model of the subunit interactions responsible for TH and TPH tetramer formation in which sequence elements in both the N-terminal and C-terminal domains are required for creating mature tetrameric enzymes. Experiments are required to determine the specific residues involved in subunit assembly and enzyme activity as well as to determine the consequence of tetramer formation in a cellular environment. proposes experiments for both TH and TPH to test the hypothesis that higher order macromolecular structures are required for the allosteric regulation of enzyme activity (aim 1). Studies are proposed that will refine the current understanding of subunit assembly by employing more selective mutagenesis and that will focus on enzyme kinetics and phosphorylation-activation of the enzymes in vitro. In aim 2, the role of higher order enzyme structures in the cellular control of hydroxylase activity will be examined by transferring selected recombinant enzymes into eukaryotic cells. This approach is designed to assess the effects of quaternary structure (ie, monomer vs tetramer) on enzyme stability, as well as on regulation by endogenous second messenger pathways. In addition to providing valuable information concerning the basic biology of hydroxylase function, the principal investigator argues that these experiments will guide future efforts in transgenic animal production. In aim 3, the applicant proposes to extend his recent observations that the related hydroxylase, tyrosinase, acts as a quinone generator to convert dopamine into a covalent suicide inhibitor of TH. This observation not only suggests a new molecular basis for dopamine neurotoxicity, but provides a novel tool for identifying important active site amino acid residues. Utilizing radiolabeled dopamine, modified residues will be identified followed by the selective mutagenesis of these residues in recombinant TH. Given the highly homologous structures of TH and TPH, the findings of these studies with the former enzyme will then be extended to the latter. Given the nature of biogenic amines in human health, argues that it is important to understand the two primary biosynthetic enzymes. The proposed experiments are designed to extend s progress in this field and to enhance our knowledge of hydroxylase function.
Keywords: enzyme activity, recombinant protein, tryptophan 5 monooxygenase, tyrosine 3 monooxygenase, allosteric site, dopamine, posttranslational modification, quinone, PC12 cell, molecular cloning, site directed mutagenesis, tissue /cell culture
Project start date: 1991-01-01
Project end date: 2002-04-30
2R01GM038931-07 (1998): $147378
MOLECULAR CHARACTERIZATION OF HUMAN TRYPTOPHAN HYDROXYLASE 2 (HTPH2)
Kent E Vrana, Professor
Pennsylvania State Univ Hershey Med Ctr, 500 University Drive, Hershey, Pa 17033-0850
Grant 3R01GM038931-18S1 from National Institute Of General Medical Sciences
Abstract: Tryptophan hydroxylase (TPH) catalyzes the initial and rate-limiting step in the biosynthesis of serotonin. As such, it has been implicated in a variety of mental health disorders. One of the goals of this long-standing R01 has been to better understand this pivotal enzyme. However, in the midst of the preceding cycle, other investigators (and current collaborators) made the seminal discovery of a new variant of this enzyme (encoded by a separate gene) that is responsible for central nervous system serotonin synthesis - TPH2. Given that the work in this field had to this point focused on the peripheral enzyme (now termed TPH1), and given the importance of CNS serotonin to health and disease, we propose shifting the emphasis of this upcoming renewal period to better understanding this novel and pivotal enzyme. Virtually nothing is known about the structure, function, and enzymology of hTPH2. Partnering with the discoverers of this new enzyme, we have established important new characterization studies on the human TPH2 (hTPH2). We are strongly positioned to pursue three specific aims in the present proposal. Specific Aim 1 will characterize the functional consequences of naturally-occurring polymorphisms in the hTPH2 gene. In the few short years since discovery of the TPH2 gene, seven different coding region polymorphisms have been described. Little is known concerning the consequences of these amino acid substitutions and this aim will address this gap in our knowledge. Aim 2 will use data we have obtained from tyrosine hydroxylase and hTPH1 to map the active site of hTPH2. These biochemical studies will provide important functional insights into this novel and important enzyme. Specific Aim 3 will explore the regulation of hTPH2 by its N-terminal regulatory domain. These experiments will contribute to our knowledge of the dynamic regulation of serotonin biosynthesis in health and disease. The novel hTPH2 gene was first described three years ago. Its discovery resolves several important discprepancies in the field of serotonin biosynthese. However, we must establish a deeper understanding of this pivotal enzyme to better appreciate its role in health and disease and to provide a basis for potential development of novel pharmacotherapeutics
Keywords: 3-(2-Aminoethyl)-1H-indol-5-ol; 5-HT; 5-Hydroxytryptamine; 5HT; Active Sites; Address; Amino Acid Substitution; Amino Acids; Anabolism; Area; Aromatic Amino Acids; Assay; Base Sequence; Bioassay; Biochemical; Biogenic Amines; Biologic Assays; Biological Assay; Biosynthetic Proteins; Catecholamines; Cell Culture Techniques; Central Nervous System; Code; Coding System; Crystallographies; Crystallography; Data; Depression; Development; Disease; Disorder; Enteramine; Enzymatic Biochemistry; Enzyme Stability; Enzymes; Enzymology; Functional RNA; Funding; Genes; Genetic Alteration; Genetic Change; Genetic Polymorphism; Genetic defect; Genetics-Mutagenesis; Goals; Grant; Health; Hippophaine; History; Human; Human, General; Hydroxylases; Image; Investigators; Kinetic; Kinetics; Knowledge; L-Phenylalanine; L-Tryptophan; L-Tyrosine; L-Tyrosine, tetrahydrobiopterin[{..}]oxygen oxidoreductase (3-hydroxylating); L-tryptophan, tetrahydropteridine[{..}]oxygen oxidoreductase (5-hydroxylating); Laboratories; Levotryptophan; Mammalian Cell; Man (Taxonomy); Man, Modern; Maps; Mental Depression; Mental Health; Mental Hygiene; Mental disorders; Mental health disorders; Mixed Function Oxidases; Mixed Function Oxygenases; Modeling; Molecular; Molecular Biology, Mutagenesis; Molecular Models; Monooxygenases; Mutagenesis; Mutation; N-terminal; NH2-terminal; NRVS-SYS; Nervous System; Nervous System, CNS; Nervous system structure; Neuraxis; Neurologic Body System; Neurologic Organ System; Non-Coding; Non-Coding RNA; Nucleic Acid Biochemistry, Molecular Modeling; Nucleotide Sequence; PC-12; PC12 Cells; Peripheral; Phenylalanine; Phenylalanine, L-Isomer; Pheochromocytoma Cell Line; Phosphorylation Site; Polymorphism (Genetics); Polymorphism, Genetic; Position; Positioning Attribute; Programs (PT); Programs [Publication Type]; Protein/Amino Acid Biochemistry, Molecular Modeling; Psychiatric Disease; Psychiatric Disorder; Psychological Health; Publishing; Recombinant Proteins; Recording of previous events; Regulation; Research Personnel; Researchers; Role; Seminal; Serotonin; Silent Mutation; Site; Structure; Structure-Activity Relationship; Substance abuse problem; Sympathins; TDO; TDO2 gene; TPH2; TYR; Testing; Time; Tph2 protein, human; Tryptophan; Tryptophan 5-monooxygenase; Tryptophan Hydroxylase; Tryptophan Monooxygenase; Tyrosine; Tyrosine 3-Monooxygenase; Tyrosine Hydroxylase; Tyrosine, L-isomer; Unspecified Mental Disorder; Variant; Variation; Work; abuse of substances; aminoacid; base; biosynthesis; chemical structure function; design; designing; disease/disorder; enzyme activity; experiment; experimental research; experimental study; gene discovery; genome mutation; human tryptophan hydroxylase 2; imaging; inhibitor; inhibitor/antagonist; insight; mental illness; molecular modeling; mutant; novel; nucleic acid sequence; para-Tyrosine; pheochromocytoma 12 cell line; polymorphism; programs; psychological disorder; research study; social role; structure function relationship; substance abuse; tryptophan 5 hydroxylase; tryptophan hydroxylase 2, human
Project start date: 1991-01-01
Project end date: 2011-02-28
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
3R01GM038931-18S1 (2010): $47985
3R01GM038931-17S1 (2009): $67856
EPIGENETIC IMPRINTING BY CHRONIC DRUGS OF ABUSE
Kent E Vrana, Elliot S. Vesell Professor And Chair
Wake Forest University 1834 Wake Forest Road Winston-salem, Nc 27106
Grant 5P50DA006634-110008 from National Institute On Drug Abuse
Abstract: These studies propose to investigate the long-term consequences of cocaine and opiate administration on molecular indices of dopaminergic function. A central problem in substance abuse research is identifying how chronic drug administration changes the brain so as to account for the long-term problems of physical dependence, psychological addiction, and tolerance. One hypothesis is that the drugs create an epigenetic imprint. That is, the chronic administration of drug alters the pattern of gene expression- the levels of RNA and protein- such that they change the state of the brain. The experiments proposed in this portion of the center application are designed to address this issue in a multi-disciplinary way by focusing on a recognized neuroanatomical substrate of substance abuse- the mesolimbic dopamine pathway. Utilizing recombinant DNA research tools developed during the last funding cycle, in combination with the facilities and expertise of both primate and rodent investigators within the Center, the following specific aims will be undertaken. In SA #1, we will test the hypothesis that chronic cocaine regulates dopaminergic gene expression in the non-human primate. Pursuing previous studies from this laboratory and others within the Center, SA #2 will examine the long-term epigenetic consequences of administration of tropane analogs with markedly different pharmacokinetics and transporter selectivity than cocaine. Further experiments are planned to identify molecular correlates for physiological and neurochemical disparities observed based on the context of drug administration. Accordingly, SA #3 will establish the relationships between epigenetic imprinting by response-dependent (self- administration) versus response-independent cocaine administration in the rodent. Finally, SA #4 will examine the hypothesis that opiate administration can alter molecular indices f dopaminergic function. These studies will continue the progress we have made in understanding how long- term drug administration affects neuronal set-points and how this might account for long-term drug abuse liabilities.
Keywords: cocaine, dopamine receptor, drug abuse, drug administration rate /duration, gene expression, pharmacogenetics, Macaca fascicularis, dopamine transporter, genetic regulation, genomic imprinting, pharmacokinetics, recombinant DNA, reinforcer, tropane, tyrosine 3 monooxygenase, animal tissue, laboratory rat, polymerase chain reaction, western blotting
Sponsored Links Excellgen http://Excellgen.com
MULTI-DISCIPLINARY TRAINING IN THE BIOLOGY OF ALCOHOLISM
Kent E Vrana, Elliot S. Vesell Professor And Chair
Wake Forest University Health Sciences Medical Center Blvd Winston-salem, Nc 27157
Grant 5T32AA007565-09 from National Institute On Alcohol Abuse And Alcoholism IRG: AA
Abstract: The rapid development of pre-clinical studies on alcohol abuse and alcoholism across many diverse disciplines presents an ever increasing training problem. Because of this multi-disciplinary data base, knowledge from molecular biology, biochemistry, neuroscience and behavior are required for appropriate synthesis and future study design. The proposed training program provides both the pre- and postdoctoral students with exposure to all facets of basic science alcohol research by a group of well-funded, committed alcohol researchers. The training faculty consists of 11 full-time members of either the Department of Biochemistry or the Department of Physiology and Pharmacology at Wake Forest University School of Medicine. Most of these members are also faculty of the interdisciplinary Neuroscience Program. The graduate trainees will have a choice between these three graduate programs. Trainee recruitment will be extensive and include an active attempt to involve minority students in the program. Given the faculty, resources and the multi-disciplinary nature of the training opportunity, be believe that each trainee will be able to continue a career in alcohol specific research following their tenure in the program. It is the primary goal of the program to assure that each trainee becomes able to understand the complex nature of alcohol s actions, with an appreciation for analysis from the molecular to the behavioral level.
Project start date: 1994-07-01
Project end date: 2004-06-30
5T32AA007565-09 (2002): $350929
SERUM BIOMARKERS OF ALCOHOL SELF-ADMINISTRATION IN NON-HUMAN PRIMATES
Kent E Vrana, Professor
Pennsylvania State Univ Hershey Med Ctr, 500 University Drive, Hershey, Pa 17033-0850
Grant 3R01AA016613-03S1 from National Institute On Alcohol Abuse And Alcoholism
Abstract: Ethanol abuse and alcoholism remain very serious societal problems. A significant problem is the inability to diagnose alcohol abuse either in the general population or within selected groups of individuals such as adolescents and the recovering alcoholic. Accordingly, this proposal seeks to develop diagnostic biomarker signatures of acute and chronic alcohol consumption for diagnosing high-risk drinking, detecting relapse to drinking, disclosing recent drinking and in high risk situations such as pregnancy. To this end, studies are proposed to examine serum proteins and protein patterns for potential signatures in a powerful non-human primate model that is not encumbered by problems of comorbid drug use, inadequate diet and unreliable assessments of drinking history. In these NIAAA-funded, ongoing, within-subject studies, monkeys have been induced to voluntarily drink large amounts of alcohol. In the course of the studies (encompassing over 100 individual animals covering years of behavior and observation), serum samples have routinely been collected and archived. Experiments are proposed to screen these samples for potential biomarkers that can then be taken forward into the human population. Serum samples from a long-standing nonhuman primate self-administration study will be used as a training set for biomarker identification using high throughput proteomics. Samples will be processedto deplete the most abundant, obscuring proteins and then subjected to 2-DIGE (2-D Fluorescence Difference In-Gel Electrophoresis) for quantitative fluorescence identification of altered serum protein expression followed by MALDI-ToF/ToF identification of protein species. Statistical validation will be conducted, in a blinded fashion, using a test set of samples from an independent colony of self-administering monkeys, which will also contain data on adolescent vulnerability. The key criteria of any putative biomarkers will be sensitivity (percentage of positive scores among drinkers) and specificity (percentage of false positives in a non-drinking population). In addition, these studies will provide initial indices of positive and negative predictive values for biomarker signatures. A clinical test for ethanol abuse and alcoholism would have many potential uses. To discover protein biomarkers of ethanol abuse and alcoholism, serum from a controlled non-human primate population self- administering ethanol will be examined by quantitative proteomic methods
Keywords: 2 Dimensional Gel Electrophoresis; 21+ years old; Absolute ethanol; Acrosomal Serine Protease Inhibitor; Acute; Address; Adolescent; Adolescent Youth; Adult; Albumins; Alcohol Drinking; Alcohol abuse; Alcohol consumption; Alcohol, Ethyl; Alcoholic; Alcoholism; Alcohols; Alpha-1 Antiproteinase; Animals; Antitrypsin; Archives; Assay; Bioassay; Biologic Assays; Biological; Biological Assay; Blinded; Blood Serum; Boozer; Chemical Class, Alcohol; Clinical; Clinical Evaluation; Clinical Testing; Cluster Analyses; Cluster Analysis; Data; Delivery of Health Care; Dependent drinker; Diagnosis; Diagnostic; Diagnostic Sensitivity; Diet; Disease; Disorder; Dose; Drug usage; ELISA; ETOH; Electrophoresis, Gel, 2-D; Electrophoresis, Gel, 2D; Electrophoresis, Gel, Two-Dimensional; Enzyme-Linked Immunosorbent Assay; Enzymes; EtOH drinking; Ethanol; Female Adolescents; Fluorescence; Funding; General Population; General Public; Gestation; Goals; Grain Alcohol; Granulocyte/Pollen-Binding Protein; Haptoglobins; Health; Health Sciences, Allied and Health Services Delivery; Healthcare Delivery; Heavy Drinking; History; Human; Human, Adult; Human, General; Immune Globulins; Immunoblotting; Immunoglobulins; Immunoglobulins / Antibodies; Individual; Institution; Intake; Interdisciplinary Research; Interdisciplinary Study; Investigators; Life; Man (Taxonomy); Man, Modern; Measurement; Methods; Methylcarbinol; Modeling; Monkeys; Multidisciplinary Collaboration; Multidisciplinary Research; PAI-3; Pathology; Pattern; Plasma Serine Protease Inhibitor; Plasminogen Activator Inhibitor 3; Plasminogen Activator Inhibitor III; Population; Predictive Value; Pregnancy; Probability; Process; Productivity; Protein C Inhibitor; Protein C Inhibitor, Activated; Proteins; Proteomics; Recording of previous events; Relapse; Research Personnel; Research Resources; Researchers; Resources; Sampling; Screening procedure; Self Administration; Self-Administered; Sensitivity and Specificity; Serine or Cysteine Proteinase Inhibitor Clade A Member 5; Serum; Serum Proteins; Siderophilin; Source; Specificity; Study Subject; Study, Interdisciplinary; Talents; Testing; Training; Transferrin; Type 3 Plasminogen Activator Inhibitor; Validation; adolescent girl; adult human (21+); alcohol ingestion; alcohol intake; alcohol problem; alcohol product use; alcohol use; alcoholic beverage consumption; alcoholic drink intake; animal resource; at-risk drinking; behavior observation; behavioral observation; biomarker; chronic EtOH drinking; chronic alcohol consumption; chronic alcohol drinking; chronic alcohol ingestion; chronic alcohol use; chronic ethanol consumption; chronic ethanol drinking; chronic ethanol ingestion; clinical test; cohort; design; designing; disease/disorder; drink heavily; drinking; drug use; ethanol abuse; ethanol consumption; ethanol drinking; ethanol ingestion; ethanol intake; ethanol product use; ethanol use; etoh use; excess alcohol consumption; excess alcohol ingestion; excess ethanol ingestion; excessive alcohol consumption; excessive alcohol ingestion; excessive alcohol intake; excessive drinking; excessive ethanol ingestion; experiment; experimental research; experimental study; extreme drinking; gel electrophoresis; gene product; hazardous alcohol use; health care delivery; heavy alcohol use; high risk; high risk drinking; indexing; juvenile; juvenile human; male; non-human primate; nonhuman primate; problem drinker; problem drinking; protein expression; research clinical testing; research study; risky drinking; screening; screenings; tool
Project start date: 2009-08-01
Project end date: 2010-07-31
Budget start date: 1-AUG-2009
Budget end date: 31-JUL-2010
3R01AA016613-03S1 (2009): $50125
1R01AA016613-01 (2007): $357059
Functional Genomics Of Cocaine Self-Administration
Kent E Vrana, Elliot S. Vesell Professor And Chair
Pennsylvania State Univ Hershey Med Ctr 500 University Drive Hershey, Pa 170330850
Grant 5R01DA013770-08 from National Institute On Drug Abuse IRG: IFCN
Abstract: Cocaine abuse remains a significant societal problem. A central theme of this research program, and the main hypothesis of this renewal application, is that chronic cocaine abuse produces significant changes in CNS gene expression that contribute to clinical issues such as tolerance, sensitization, physical dependence, craving, and withdrawal. Preliminary findings have added to the growing database on cocaine-responsive gene expression in the central nervous system. Experiments are proposed, within the present application, that extend these findings to identify those genes whose expression is altered following an enforced abstinence from cocaine self-administration. To accomplish this, a new behavioral paradigm has been developed in which a binge pattern of cocaine self-administration in rats, followed by a period of abstinence, produces a behavioral sensitization that displays some of the hallmarks of the addictive process in humans. This powerful behavioral model will be examined to identify those genes exhibiting persistent changes in gene expression following cessation of drug administration. The first series of experiments will examine the expression of known cocaine-responsive genes to see if they correlate with the behavioral sensitization. The studies will focus on the nucleus accumbens (core and shell), medial prefrontal cortex, hippocampus, and amygdala as neuroanatomical substrates of the behavioral perturbation. In addition, subsequent studies will concentrate on establishing the temporal course of gene expression to (a) establish the limits of the alterations (how long will they last?), and (b) determine the time at which the changes first become manifest (before or during the abstinence period?). The second series of studies will use new Affymetrix GeneChip technologies (14,280 genes at a time) to identify families of genes that may be coordinately regulated and to identify novel targets of cocaine s effects. The proposed experiments will continue to contribute to our understanding of the role of genomics in establishing and maintaining cocaine addiction.
Keywords: behavioral habituation /sensitization, cocaine, drug abuse, functional /structural genomics, pharmacogenetics, self medication, substance abuse related behavior, amygdala, gene expression, hippocampus, neuropharmacology, nucleus accumbens, prefrontal lobe /cortex, behavioral /social science research tag, laboratory rat, microarray technology
Project start date: 2000-09-30
Project end date: 2008-07-31
5R01DA013770-08 (2006): $257421
5R01DA013770-07 (2005): $263616
5R01DA013770-06 (2004): $234274
Sponsored Links Excellgen http://Excellgen.com
2R01DA013770-04A1 (2003): $287917
FUNCTIONAL GENOMICS OF COCAINE SELF ADMINISTRATION
Kent E Vrana, Elliot S. Vesell Professor And Chair
Physiology And Pharmacologywake Forest University Health Sciences
medical Center Blvd
winston-salem, Nc 27157
Grant 5R01DA013770-02 from National Institute On Drug Abuse IRG: ZDA1
Abstract: Applicant´s ) Cocaine and crack addiction remains a significant medical and social problem. While the dopamine transporter is thought to be the primary site of action for the acute reinforcing effects of cocaine, the post-synaptic consequences of cocaine use and the neurobiological mechanisms that subserve the addictive process remain to be confirmed. The identification of the genetic components underlying cocaine addiction is a critical step in identifying potential targets for therapeutic intervention. The central hypothesis of this application is that chronic drug abuse produces a metastable epigenetic imprint that may contribute to clinical issues such as tolerance, physical dependence, and withdrawal. This application proposes to use multiplex DNA hybridization arrays to examine the interface of functional genomics and behavior. - In Specific Aim # 1, DNA hybridization arrays will be used to profile the CNS epigenetic imprint induced by cocaine self-administration in rats. This will be accomplished using commercially available arrays as well as custom arrays, imprinted at this institution and designed to test specific hypotheses regarding cocaine abuse. These will then be followed by studies in Specific Aim #2 that examine a new binge abstinence model of cocaine administration. This model recapitulates several key features of human cocaine abuse (specifically, the progressive loss of behavioral control). Finally, studies at the end of the funding period (Specific Aim #3) will establish the time course for gene expression changes following cessation of cocaine self-administration. These last experiments will provide very important insights into the stable changes in gene expression that survive long-term cessation of the drug, while simultaneously identifying new genes whose expression is altered during the withdrawal period. In addition to the specific proposed experiments, efforts will be coordinated with Emory University, during the funding period, to establish a central repository for array data for general access by the NIDA research community (www.arraydata.org) as well as a tissue/RNA bank. The results of the studies described within the present application should provide a wealth of information concerning functional genomic contributions to the cocaine addiction process
Keywords: cocaine, drug abuse, functional genomics, genomic imprinting, pharmacogenetics dopamine receptor, drug tolerance, drug withdrawal, gap junction, gene expression, limbic system, membrane channel, self medication laboratory rat, microarray technology
Project start date: 2000-09-30
Project end date: 2003-05-31
5R01DA013770-02 (2001): $325219
1R01DA013770-01 (2000): $355457
SERUM BIOMARKERS OF ALCOHOL SELF-ADMINISTRATION IN NON-HUMAN PRIMATES
Kent E Vrana, Professor
Pennsylvania State Univ Hershey Med Ctr, 500 University Drive, Hershey, Pa 17033-0850
Grant 5R01AA016613-03 from National Institute On Alcohol Abuse And Alcoholism
Keywords: 2 Dimensional Gel Electrophoresis; 21+ years old; Absolute ethanol; Acrosomal Serine Protease Inhibitor; Acute; Address; Adolescent; Adolescent Youth; Adult; Albumins; Alcohol Drinking; Alcohol abuse; Alcohol consumption; Alcohol, Ethyl; Alcoholic; Alcoholism; Alcohols; Alpha-1 Antiproteinase; Animals; Antitrypsin; Archives; Assay; Bioassay; Biologic Assays; Biological; Biological Assay; Blinded; Blood Serum; Boozer; Chemical Class, Alcohol; Clinical; Clinical Evaluation; Clinical Testing; Cluster Analyses; Cluster Analysis; Data; Delivery of Health Care; Dependent drinker; Diagnosis; Diagnostic; Diagnostic Sensitivity; Diet; Disease; Disorder; Dose; Drug usage; ELISA; ETOH; Electrophoresis, Gel, 2-D; Electrophoresis, Gel, 2D; Electrophoresis, Gel, Two-Dimensional; Enzyme-Linked Immunosorbent Assay; Enzymes; EtOH drinking; Ethanol; Female Adolescents; Fluorescence; Funding; General Population; General Public; Gestation; Goals; Grain Alcohol; Granulocyte/Pollen-Binding Protein; Haptoglobins; Health; Health Sciences, Allied and Health Services Delivery; Healthcare Delivery; Heavy Drinking; History; Human; Human, Adult; Human, General; Immune Globulins; Immunoblotting; Immunoglobulins; Immunoglobulins / Antibodies; Individual; Institution; Intake; Interdisciplinary Research; Interdisciplinary Study; Investigators; Life; Man (Taxonomy); Man, Modern; Measurement; Methods; Methylcarbinol; Modeling; Monkeys; Multidisciplinary Collaboration; Multidisciplinary Research; PAI-3; Pathology; Pattern; Plasma Serine Protease Inhibitor; Plasminogen Activator Inhibitor 3; Plasminogen Activator Inhibitor III; Population; Predictive Value; Pregnancy; Probability; Process; Productivity; Protein C Inhibitor; Protein C Inhibitor, Activated; Proteins; Proteomics; Recording of previous events; Relapse; Research Personnel; Research Resources; Researchers; Resources; Sampling; Screening procedure; Self Administration; Self-Administered; Sensitivity and Specificity; Serine or Cysteine Proteinase Inhibitor Clade A Member 5; Serum; Serum Proteins; Siderophilin; Source; Specificity; Study Subject; Study, Interdisciplinary; Talents; Testing; Training; Transferrin; Type 3 Plasminogen Activator Inhibitor; Validation; adolescent girl; adult human (21+); alcohol ingestion; alcohol intake; alcohol problem; alcohol product use; alcohol use; alcoholic beverage consumption; alcoholic drink intake; animal resource; at-risk drinking; behavior observation; behavioral observation; biomarker; chronic EtOH drinking; chronic alcohol consumption; chronic alcohol drinking; chronic alcohol ingestion; chronic alcohol use; chronic ethanol consumption; chronic ethanol drinking; chronic ethanol ingestion; clinical test; cohort; design; designing; disease/disorder; drink heavily; drinking; drug use; ethanol abuse; ethanol consumption; ethanol drinking; ethanol ingestion; ethanol intake; ethanol product use; ethanol use; etoh use; excess alcohol consumption; excess alcohol ingestion; excess ethanol ingestion; excessive alcohol consumption; excessive alcohol ingestion; excessive alcohol intake; excessive drinking; excessive ethanol ingestion; experiment; experimental research; experimental study; extreme drinking; gel electrophoresis; gene product; hazardous alcohol use; health care delivery; heavy alcohol use; high risk; high risk drinking; indexing; juvenile; juvenile human; male; non-human primate; nonhuman primate; problem drinker; problem drinking; protein expression; research clinical testing; research study; risky drinking; screening; screenings; tool
Project start date: 2007-01-01
Project end date: 2010-12-31
Budget start date: 1-JAN-2009
Budget end date: 31-DEC-2010
5R01AA016613-03 (2009): $329118
Molecular Characterization Of Human Tryptophan Hydroxylase 2 (hTPH2)
Kent E Vrana, Elliot S. Vesell Professor And Chair
Pennsylvania State Univ Hershey Med Ctr 500 University Drive Hershey, Pa 170330850
Grant 2R01GM038931-15A1 from National Institute Of General Medical Sciences IRG: ZRG1
Project start date: 1991-01-01
Project end date: 2011-02-28
2R01GM038931-15A1 (2007): $189429
MOLECULAR CHARACTERIZATION OF BIOGENIC AMINE SYNTHESIS
Kent E Vrana, Elliot S. Vesell Professor And Chair
Physiology And Pharmacologywake Forest University
1834 Wake Forest Road
winston-salem, Nc 27106
Grant 3R01GM038931-06S1 from National Institute Of General Medical Sciences IRG: NEUC
Project start date: 1991-01-01
Project end date: 1998-04-30
3R01GM038931-06S1 (1998): $12281
DRUG INDUCED EPIGENETIC CHANGES IN DA RECEPTORS MRNA LEVELS
Kent E Vrana, Elliot S. Vesell Professor And Chair
Wake Forest University 1834 Wake Forest Road Winston-salem, Nc 27106
Grant 5P50DA006634-07S10008 from National Institute On Drug Abuse
Keywords: dopamine receptor, drug abuse, messenger RNA, pharmacokinetics, reinforcer, brain mapping, cocaine, gene expression, heroin, receptor binding, self medication, behavioral /social science research tag, laboratory rat, polymerase chain reaction
Project start date: 1997-09-01
Project end date: 1999-01-31
Sponsored Links Excellgen http://Excellgen.com