The Characterization of a Human Nitric Oxide Synthase I Promoter Complex PDF Download

Author: Terrie K. Rife
Publisher:
ISBN:
Category :
Languages : en
Pages : 324

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Author:
Publisher:
ISBN:
Category :
Languages : en
Pages :

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Publisher: Academic Press
ISBN: 0080536379
Category : Science
Languages : en
Pages : 375

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Although the function of nitric oxide in a regulatory capacity in the central and peripheral nervous system is widely recognized, the full scope of its actions and its interrelationships with other classes of regulatory molecules is just beginning to be comprehended. This volume contains a number of sophisticated and advanced methods essential for exploring the activity of nitric oxide in the brain. It will be a valuable tool for the established investigator and for those just entering the field. Comprehensive protocols included for detection of NO and related compounds by chemical, immunohistochemical, and in situ hybridization techniques Newly developed methods for the purification of neuronal and endothelial NO synthase, production of monoclonal antibodies to NO synthase, molecular cloning and expression of NO synthesis, and control of NO synthase gene expression Assessment of NO-mediated functions in neurons, central nervous system, cerebral circulation, synaptic transmission, and vascular tone Calcium imaging by confocal microscopy, evaluation of the effect of NO on iron metabolism, and detection of heme oxygenase-1 and -2 message level and distribution

Author: Fotula Karantzoulis-Fegaras
Publisher:
ISBN:
Category :
Languages : en
Pages : 0

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Understanding transcription initiation of the endothelial nitric oxide synthase (eNOS) gene appears pivotal to gaining a comprehensive view of NO biology in the blood vessel wall. The present thesis focused upon a detailed dissection of the functionally important cis-DNA elements and the multiprotein complexes implicated in the cooperative control of the constitutive expression of the human eNOS gene in vascular endothelium. Three tightly clustered cis-regulatory regions were identified in the proximal enhancer of the TATA-less eNOS promoter using deletion analysis and linker-scanning mutagenesis: positive regulatory domain I (PRD I) -104/-95 relative to transcription initiation), PRD II (-144/-115) and PRD III (-44/-25). The nucleoprotein complexes that form upon these regions in endothelial cells contained Ets family members, Sp1 and variants of Sp3, MAZ and YY1. Functional domain studies in 'Drosophila' Schneider cells and endothelial cells revealed examples of positive and negative protein-protein cooperativity involving Sp1, variants of Sp3, Ets-1, Elf-1 and MAZ. The multiprotein complexes formed on the activator recognition sites within this region of the human eNOS promoter identify the existence of a constitutively active enhanceosome in vascular endothelium. Moreover, 'in vitro' DNase I footprinting analyses and nuclear run-off studies also indicate their important contributions to preinitiation complex formation. Surprisingly, episomal expression studies performed in endothelial and non-endothelial cell types revealed that eNOS promoter/reporter constructs lacked cell-specificity, suggesting that there exist cell-specific mechanisms that repress native eNOS gene expression in non-expressing cell types. We posited that DNA methylation played a role in this cell-specific expression. Sodium bisulfite genomic sequencing revealed a different methylation pattern of the eNOS promoter in endothelial versus non-endothelial cells. Transient transfection of 'in vitro' methylated promoter/reporter constructs revealed a possible role for methylation in transcriptional repression. DNA hypomethylation studies performed with 5-azacytidine, resulted in increased expression of eNOS mRNA in cell types that do not normally express the eNOS mRNA transcript. The role of epigenetic regulation in cell-specific eNOS gene expression is a novel finding in the vascular endothelium. We have identified novel concepts with respect to protein-DNA interactions, pre-initiation complex formation and chromatin-based epigenetic mechanisms in vascular endothelium.

Author: Gregory Thomas Hartt
Publisher:
ISBN:
Category : Gene expression
Languages : en
Pages :

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Abstract: Nitric oxide is a gaseous, free radical molecule that functions in the nervous system as an atypical neurotransmitter, second messenger, vasodilator, and potent neurotoxin, depending on cell type and level of expression. Physiologically, nitric oxide is thought to mediate long-term potentiation, the cellular correlate of learning and memory formation, as well as synaptic plasticity and remodeling. Under pathologic conditions such as cerebral ischemia, NOS1 overproduces nitric oxide, which is a key mediator of excitotoxic cell death and neurodegeneration. Our laboratory discovered that multiple, individually functioning promoters (5'1 and 5'2) regulate human NOS1 gene expression. The present work describes the discovery and cloning of a novel human NOS1 promoter, 5'3, and the demonstration of its unique, developmentally regulated expression pattern in the central nervous system. Promoter-specific alternative splicing contributes to NOS1 mRNA diversity in both the 5' untranslated and coding regions of the gene. We generated several lines of transgenic mice that express reporter genes under the control of separate human NOS1 promoter complexes (PR(5'1+5'2) and PR(5'3+5'4)), and studied how these alternate promoters contribute to NOS1 expression in various physiologic and pathophysiologic states in vivo. To this end, transgene expression is documented throughout normal development, which demonstrates overlapping but distinct patterns of promoter use that contribute to the overall recapitulation of endogenous NOS1 gene expression. In a murine model of striatal neurotoxicity, systemic methamphetamine administration results in promoter-specific transcriptional activation in the transgenic mice. NOS1 enzymatic activity is stimulated in motor neuron cell bodies after peripheral transection of the facial nerve; however, the transgenic NOS1 promoter complexes are insufficient to recapitulate NOS1 activation in this model. These findings help clarify how multiple promoters and mRN A diversity contribute to the complex regulation of the human NOS1 gene, and further our general understanding of transcriptional regulation of gene expression via alternate promoters.

Author: Derek Colin Newton
Publisher: National Library of Canada = Bibliothèque nationale du Canada
ISBN: 9780612917705
Category :
Languages : en
Pages : 644

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Protein synthesis is the ultimate goal of gene expression and a key control point for regulation. In particular, it enables the cell to rapidly manipulate protein production without new mRNA synthesis, processing or export. The human neuronal nitric oxide synthase (nNOS) gene is an excellent model to study the regulated translation of a mRNA to protein. Numerous and distinct nNOS mRNAs are generated from a single gene locus as a result of alternate promoter usage and alternate splicing patterns. A comprehensive analysis of the structure of neuronal NOS (nNOS) mRNA species revealed NOS1 to be one of the most structurally diverse human genes described to date in terms of promoter usage. Unique exon 1 variants are utilized for transcript initiation in diverse tissues and this dependence on unique genomic regions to control transcription initiation in a cell-specific fashion burdens the transcripts with complex 5'-mRNA leader sequences. These highly structured nNOS mRNA 5'-untranslated regions (5'-UTRs) were demonstrated to have profound effects on translation in both in vitro assays and cells and to contain cis-RNA elements that modulate translational efficiency in response to changes in cellular phenotype. In addition to promoter diversity, the human nNOS gene contains an alternately spliced (AS) exon present within the 5' -UTR. Alternative splicing events that do not affect the open reading frame are distinctly uncommon in mammals; therefore we assessed its functional relevance. Transient and stable transfection methods revealed that this alternatively spliced exon acts as a potent translational repressor. Hence a unique splicing event within a 5'-UTR is demonstrated to introduce a translational control element. Importantly, this represented a newer model for the translational control in mammalian cells. Mutational analysis identified a putative stem-loop structure within this exon that acts as the functional cis-element. Subsequent RNA-EMSA analyses revealed that a specific cytoplasmic RNA-binding complex interacts with this motif. An affinity purification scheme was developed and the cytoskeletal proteins actin and alpha-actinin were identified as components of the ribonucleoprotein complex associated with this exon. This intriguing finding indicates that this exon acts as a subcellular localization signal for select nNOS mRNAs.

Author: Seyed Soheil Saeedi Saravi
Publisher: BoD – Books on Demand
ISBN: 953513163X
Category : Science
Languages : en
Pages : 250

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Nitric Oxide Synthase - Simple Enzyme-Complex Roles provides information on nitric oxide synthase, a biomolecule of key importance for the different biological systems, including central and peripheral nervous, cardiovascular, and reproductive systems. With recent links to the role of nitric oxide in the reactions that can impact cell signaling, and discoveries surrounding the complex role of nitric oxide synthase that have increased research attention across the fields of cell and molecular biology, physiology, pharmacology, toxicology, neuroscience, cardiology, urology, and endocrinology, this book tries to provide a comprehensive overview of biology/pathobiology of nitric oxide synthases and a perspective from possible therapeutic indication of the enzyme inhibitors.

Author: Ernst van Faassen
Publisher: Elsevier
ISBN: 0080489591
Category : Science
Languages : en
Pages : 444

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Radicals for Life: the Various Forms of Nitric Oxide provides an up-to-date overview of the role of nitrosocompounds and nitrosyl-iron complexes in physiology. Nitrosocompounds can be considered as stabilised forms of nitric oxide, one of the most important regulatory molecules in physiology today. Many nitrosocompounds share some of the physiological functions of nitric oxide, and may be formed inside living organisms. This is the first book to be published that is dedicated to the role of such nitrosocompounds in physiology, with particular emphasis on the nitrosocompounds that are endogenously formed in higher organisms and humans. Points of discussion include: physical and chemical properties of the compounds, the main chemical pathways in vivo, as well as the physiological effects that have been recognised to date. Each of the nineteen chapters is written by distinguished specialists in the field, well known for their original and important contributions to the subject. Also included are results from a wide range of studies in vitro, in cell cultures, animal models and human volunteers. Examples of alternative forms of nitric oxide, with special emphasis on their protective role against widespread human diseases like atherosclerosis, Alzheimer's disease, diabetes, sexual dysfunction, and renal insufficiency to stroke and ischemia are also included. First monograph to consider and provide an overview of endogenous nitrosocompounds and nitrosyl-iron complexes Extensive bibliographic references, written by specialists of human physiology Providing high scientific quality with a focus on implications for human diseases

Author:
Publisher:
ISBN:
Category : Medicine
Languages : en
Pages : 1844

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Author:
Publisher:
ISBN:
Category : Dissertations, Academic
Languages : en
Pages : 882

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