1985 — 2001 |
Shih, Jean C |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Protein(S) Involved in Neurotransmission @ University of Southern California
5-hydroxytryptamine (5-HT, serotonin) and its receptors have been implicated in the etiology and therapeutic treatment of human mental depression. A basic knowledge of 5-HT receptors will help to better understand the molecular mechanism of 5-HT mediated neural function and will provide insights into the molecular basis of mental depression. The objectives of this project are to investigate the structure and function relationships, the genomic organization and the regulation of human 5-HT-2 receptor(s). These objectives will be accomplished by using the rat brain 5-HT-2 receptor cDNA cloned recently. The specific aims of the proposed study are outlined below. 1. To clone 5-HT-2 receptor and related cDNAs from human brain. Various 5-HT-2 receptor subtypes and related cDNA clones will be isolated and sequenced. The pharmacological profiles and the second messenger systems linked to the expressed receptors (phosphoinositol (PI) turnover, CA++ release, or adenylate cyclase) will be characterized. 2. To map the chromosomal location of the 5-HT-2 receptor gene. This aim will be accomplished by somatic cell mapping and in situ hybridization of the chromosome. 3. To isolate human genomic 5-HT-2 receptor clones. 4. To construct the restriction map and to sequence human 5-HT-2 receptor genomic clones. 5. To identify and to analyze promoter regions of this gene. This aim will be accomplished by RNase protection, primer extension, and chloramphenicol acetyltransferase (CAT) reporter gene assay. The cis- elements, trans-acting factors and the functions of these elements will be systematically analyzed. 6a. To investigate if 5-HT-2 receptor gene is regulated by protein kinase C (PKC) and/or protein kinase A (PKA). The effects of TPA (12-0- tetradecanoyl-phorbol-14 acetate, activator of PKC) on the 5-HT-2 receptor gene expression will be studied by using CAT report gene assay and footprinting analysis. The ability of 1-(5-isoquinolylsulfonyl)-2- methylpiperazine (H7) to block TPA activation will be studied. Similar studies will be performed using activators of PKA such as forskolin and 8- bromodibutyryl cAMP. 6b. To investigate if antagonist induced 5-HT-2 receptor down regulation is at the transcriptional level.
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0.958 |
1985 — 2013 |
Shih, Jean Chen |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Two Types of Monoamine Oxidase @ University of Southern California
gas chromatography mass spectrometry; high performance liquid chromatography; laboratory mouse; site directed mutagenesis
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0.958 |
1989 — 1998 |
Shih, Jean C |
K05Activity Code Description: For the support of a research scientist qualified to pursue independent research which would extend the research program of the sponsoring institution, or to direct an essential part of this research program. |
Molecular Studies of Monoamine Oxidases @ University of Southern California
The overall objective of this project is to understand the molecular basis of two types of monoamine oxidase (MAO A and B) at the gene level. MAO is an important enzyme in catecholamine metabolism. Abnormal levels of MAO activity have been shown in a number of mental disorders. With the full-length human liver MAO A and B cDNAs cloned in this laboratory, the genomic organizations and the expressions of these genes will be investigated. These studies have great importance for both basic and clinical research. The specific aims of this five-year Research Scientist Award application are described below. Using the established bacterial and mammalian cell expression plasmid vectors, the cloned human liver MAO A and B cDNAs will be expressed and the catalytic properties of the expressed proteins will be characterized. Using Northern blot analysis with MAO A and B cDNA as probes, the correlation among the expressions of these genes at the levels of mRNA, protein and catalytic activities in various human tissues will be examined. This study will indicate whether the tissue specificity of MAO A and B is controlled at the level of transcription or translation. Using Southern blot analysis of cells containing human chromosomes - mouse DNA hybrid as well as in situ hybridization, the chromosomal location of human liver MAO A and B genes will be determined. This fundamental knowledge is essential for studying the role of MA0 genes in mental disorders. The genomic DNAs For MAO A and B will be analyzed. Several human genomic libraries (cosmic and LAMBDA) will be screened using MAO A and B cDNA probes, the genomic DNAs encoding human liver MAO A and B genes will be cloned. The restriction maps of the genomic MAO A and B clones will then be constructed. Further, the MAO A and B genes will be sequenced by the chain termination method, and their promoter regions will be analyzed. The heterogeneity of MAO A and B will be investigated. MAO A, B and related cDNAs will be cloned from other human tissues, and their DNA and amino acid sequences will be compared. Thus, the similarities and differences of MAO A (or B) from various tissues will be clearly understood. This information will provide a conclusive answer on the validity of using platelet MAO B as a marker for brain MAO B and will have significant implications on future clinical studies.
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0.958 |
2004 — 2008 |
Shih, Jean Chen |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Transcriptional Regulation of Monoamine Oxidase A @ University of Southern California
DESCRIPTION (provided by applicant): The long-term objective of this project is to understand the role of monoamine oxidase A (MAO A) in mental disorders. MAO A is the key enzyme, which degrades serotonin (5-HT). This application focuses on the transcriptional regulation of MAO A. The new information obtained will provide new insights on the molecular basis of diseases associated with abnormal levels of 5-HT. It will also help develop a new series of MAO A inhibitors targeted at the transcriptional level. We have recently cloned a novel Repressor R1 specific for MAO A promoter. We have also found that steroids (androgen and glucocorticoid) activate the MAO A gene expression. With these new findings we hypothesize that the interactions among R1, Spl, a functional polymorphism, 30 bp variable number of tandem repeat (VNTR), steroid receptors and other associated proteins play important roles in the transcriptional regulation of MAO A. Specific aims are: 1. To investigate if Repressor R1 binds to Spl site or the GC rich region by site-directed mutagenesis, luciferase functional assay and gel shift assay. Human neuroblastoma (SK-N-BE (2)-C) and androgen dependent prostate carcinoma (LNCaP) cell lines will be used. The role of Cysteines on R1 function will be studied. The interaction between endogenous R1 and native MAO A promoter will be studied by chromatin immunoprecipitation (CHIP) assay. The biological function of R1 will be studied by RNA interference (RNAi) and serum starvation induced apoptosis. 2. To investigate the dynamic intracellular location of R1 protein in living cells by fusion R1 with enhanced Green Fluorescence Protein (eGFP). The brain regional distribution of R1 protein and mRNA will be studied by immunohistochemistry and in situ hybridization, respectively. They will be correlated with that of MAO A and B. The embryonic and postnatal development of R1 will be studied. 3. The effect of VNTR on R1 function and MAO A promoter activity will be investigated. Repressor R1 interacting proteins in SK-N-BE (2)-C cells will be co-immuno-precipitated using specific polyclonal R1 antibody developed in this laboratory.The proteins associated with R1 will be identified by their partial amino acid sequences by LC/MS/MS mass spectrometry. The validity and the function of these R1 interacting proteins will be investigated. 4. To identify the functional glucocorticoid (androgen) response element and to study the direct effect of AR/GR on MAO A promoter activity. The effect of VNTR on AR/GR activation of MAO A promoter will be studied. 5. To investigate if AR/GR indirectly activates MAC) A gene expression by interacting with Spl and other coactivators. The interactions among AR, GR, Spl and R1 on MAO A gene expression will be studied by luciferase assay (promoter activity), Northern blot (mRNA), Western blot (protein) and catalytic activity. AR (PC-3) and GR (Cos 7) negative cell lines will also be used. Co-regulators interacting with APJGR during agonist stimulated or basal state will be co-immuno-precipitated by AR or GR specific antibody. The validity and the function of identified protein(s) will be investigated.
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0.958 |