Browsing by Subject "Department of Pharmacology"

Now showing 1 - 8 of 8
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    Creation of a DNA Substrate for the Investigation of DNA-Protein Crosslinks
    (2010-04-21) Anwah, Ifeanyirochukwu G.
    Bialkylating agents form DNA-protein crosslinks (DPCs) as a side reaction. Bialkylating agents belong to a class of drugs used to treat cancer that function by forming DNA-DNA crosslinks and interfering with DNA function. To a lesser extent, they also form DNA-protein crosslinks (DPCs). DPCs are know to be harmful to the cell, but they have not been well-studied. The aim of our research was to study these DPCs and better understand their mechanism of action. A better understanding of the function of DPCs could one day contribute to the development of novel and more efficacious drugs used to treat cancer.
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    The Identification of Girk Channel Domains that Facilitate Rapid and Efficient Coupling to G Protein- Coupled Receptors
    (2011-04-13) Young, Daniele
    G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels constitute one subfamily of potassium-selective ion channels that regulate neuronal activity and heart rate. GIRK channels have been implicated in many biological processes, including pain perception, learning and memory, food intake, and reward. Structurally, GIRK channels consist of 4 subunits, able to form homotetramers or heterotetramers within the cell membrane. Activation of these channels occurs through a signal transduction cascade originating from ligandstimulated G-protein coupled receptors (GPCRs). This results in hyperpolarization of the cell membrane. With the variety of biological processes GIRK channels are involved in, understanding the interaction of the GPCR signaling cascade with GIRK channels could provide a beneficial therapeutic target. Using a variety of molecular biology techniques, I synthesized various chimeric proteins to investigate the GIRK channel and GPCR relationship, specifically focusing on amino acid residues promoting the coupling of GIRK1 with the GABAB receptor. Functional and biochemical assays in native systems suggest the Cterminal region and pore residue of the GIRK1 subunit are necessary to mediate a large receptor-induced response from the GIRK channel. Investigating this phenomenon further can determine how the identified GIRK1 channel domains are mediating efficient GABAB receptor coupling.
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    Intracellular trafficking of opioid receptor subtypes in response to ligand activation
    (2012-04-18) McGarrah, Patrick
    Opioids are currently the most powerful medications available for the treatment of severe pain. Unfortunately, their strong analgesic capabilities are complicated by the risk of tolerance, dependence, and abuse. Previous studies have modeled drug addiction as a pathological form of experienced-based learning. Opioids have profound effects on the stability of dendritic spines, which are believed to be important in learning and memory. The internalization of opioid receptors following ligand binding positively influence the stability of dendritic spines. The present study examined whether different opioid receptor subtypes could be induced to internalize following administration of a ‘non-internalizing’ agonist, such as morphine.
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    Investigating the Induction and Purification of OGG1 Protein and Its Role in DNA-Protein Cross-Linking
    (2010-04-21) Nguyen-Tran, Thuy Duong
    DNA-protein cross-links (DPC’s) occur when a protein reacts with a DNA strand. DPC’s can lead to cell death if it is not repaired due to its potential to interfere with cellular processes such as DNA replication and transcription because it causes a bulky distortion in the DNA double helix. Various chemical agents, such as 1,2,3,4-Diepoxybutane (DEB), are known to induce DPC’s because of their alkylating functions. When uncontrollably created in cells, DPC’s have a harmful effect, but they can have a therapeutic function if they are specifically induced in cells and controlled. Thus, with its potential lethalness to cells, DPC’s have been studied as a possible cancer treatment by trying to target its production in tumor cells and observing its biological activity. To better understand, DPC’s, this project focused on the creation of DPC’s using 8-oxoguanine glycosylase (OGG1) and the OG oligonucleotide duplex (OG). Specific focus was placed on the induction and purification of the OGG1 protein and cross-linking it to OG.
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    The PKC Inhibitor Gö 6976 Blocks C-Type Natriuretic Peptide Activation of Guanylyl Cyclase B
    (2009-10-07) Lou, Xiaoying
    This study characterizes the effects of the widely used protein kinase C inhibitor, Gö 6976, on NPR-B guanylyl cyclase activity as a means to identify its inhibitory mechanisms.
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    The Protein Kinase C Inhibitor Gö6976 Blocks C-Type Natriuretic Peptide Activation of Guanylyl Cyclase B
    (2010-04-21) Lou, Xiaoying
    C-type natriuretic peptide (CNP) activates the transmembrane guanylyl cyclase natriuretic peptide receptor-B (NPR-B/GC-B), which stimulates cGMP synthesis and mediates long bone growth, vasorelaxation, and axonal guidance. This study characterizes the effects of the widely used protein kinase C inhibitor, Gö6976, on NPR-B guanylyl cyclase activity. In membrane guanylyl cyclase assays, Gö6976 inhibited CNP-dependent guanylyl cyclase activity, but had no effect on detergent dependent guanylyl cyclase activities, suggesting that the effect of Gö6976 is on the CNP-dependent activation process, not on the formation of the catalytic site. Gö6976 inhibited mutant forms of NPR-B lacking known phosphorylation sites in a manner similar to in the wild type receptor, consistent with a process that does not require changes in receptor phosphorylation status. Nanomolar concentrations of Gö6976 inhibited NPR-B activity in 293 cells sensitive to phorbol ester-dependent inhibition of NPR-B (293PMA), whereas micromolar amounts of Gö6976 were required to produce a less marked effect on NPR-B activity in 293 cells that are moderately sensitive to PMA inhibition (293T), and no significant inhibition by Gö6976 was observed in a line of 293 cells that are not sensitive to phorbol ester-dependent inhibition (293neo). The differential inhibition of NPR-B activity in these cell lines suggests that cellular environment plays a critical role in the mechanism of Gö6976-dependent inhibition of NPR-B. Finally, direct addition of Gö6976 to crude 293T cell membrane also reduced CNP-dependent guanylyl cyclase activity, indicating that the preservation of cellular machinery is not required for the inhibitory process.
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    REGULATION OF GUANYLYL CYCLASE-B: ROLES OF ATP AND P-SITE INHIBITORS
    (2011-04-13) Lou, Xiaoying
    C-type natriuretic peptide (CNP) activates the transmembrane guanylyl cyclase natriuretic peptide receptor-B (NPR-B/GC-B), which stimulates cGMP synthesis and mediates long bone growth, vasorelaxation, and axonal guidance. Genetic mutations that disrupt normal signaling through this pathway lead to human skeletal over- and under-growth. Currently, the role of ATP in receptor activation is controversial. Although ATP is not required for initial activation, it may stabilize the catalytic domain at longer time points and increase its affinity for GTP. Enzymatic time course experiments conducted in crude membranes indicated that 1mM ATP increases guanylyl cyclase activity 2-3 fold when measured at high (1mM) GTP concentrations but increases activity 10-fold at low (0.1mM) GTP concentrations. Moreover, receptor activity declines at a greater rate in the absence of ATP than in its presence at 0.1mM [GTP] versus 1mM [GTP]. We propose that product inhibition, the binding of purine nucleotides and pyrophosphate to the catalytic domain, explains the observed receptor deactivation. Additional experiments are underway to characterize the roles of P-site inhibitors on GC-B activity, which may provide new insights into the catalytic mechanism of guanylyl cyclase receptors.
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    Role of CD38/cyclic-ADP-ribose in Human Asthma
    (2009-04-08) Smelter, Dan F.
    CD38 is a cellular protein found throughout the mammalian body. It is involved in calcium signaling and innate immunity. Its expression is augmented by the presence of multiple contractile agonists that occur in the development of asthma, and it is implied that CD38 expression is involved in the pathogenesis of asthma. However, CD38's role in human asthma has not been described. In my studies, I wanted to determine whether CD38 expression is involved in human asthma. To test this, I studied the effects of TNF-alpha-induced augmented CD38 expression in airway smooth muscle cells obtained from asthmatics compared to non-asthmatics. Also, I tested the intracellular calcium response to contractile agonists in cells from asthmatics and non-asthmatics following exposure to TNF-alpha.