Browsing by Author "Dr. Robert B. Rose, Committee Member"

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    Protein Kinase A Regulates beta-2 Integrin Avidity Activation and Subsequent Neutrophil Activation via Modulation of Myosin Light Chain Kinase.
    (2006-04-11) Chilcoat, Clayton Douglas; Dr. Robert B. Rose, Committee Member; Dr. Samuel L. Jones, Committee Co-Chair; Dr. Scott M. Laster, Committee Member; Dr. Edward A. Havell, Committee Member; Dr. Wayne A. Tompkins, Committee Co-Chair
    β2 integrins are adhesion molecules on the surface of neutrophils. Avidity activation of β2 integrins includes transportation of pre-formed integrins to the cell surface and a conformational change in the integrin to a high-binding state. Upon binding ligand, β2 integrins initiate a signaling cascade that results in activation of the neutrophil to a pro-inflammatory state, and the inhibition of this signal can prevent further activation of the neutrophil. cAMP and it effector protein kinase A (PKA) exert a generally inhibitory effect upon neutrophil activation. PKA has been shown to inactivate myosin light chain kinase (MLCK). Myosin light chain (MLC) phosphorylation is crucial for actin-myosin complex formation, which is required for stability and contraction of the actin cytoskeleton in neutrophils as well as β2 integrin-dependent adhesion. We hypothesize that the inhibitory effect of PKA upon neutrophils is due to inhibition of β2 integrin avidity activation resulting in the subsequent inhibition of neutrophil activation. Furthermore we hypothesize that the effect of PKA upon β2 integrin avidity activation is mediated through PKA's effect upon MLCK. We demonstrate that inhibition of PKA induces β2 integrin-dependent adhesion and that augmentation of cAMP prevented β2 integrin-dependent adhesion and subsequent respiratory burst activity. Further, we demonstrate via flow cytometric detection of antibodies directed against β2 integrins that pharmacologic inhibition of PKA activity results in overall increased β2 integrin expression on the neutrophil surface, as well as increased expression of the activated form of the integrin. This upregulation and activation of β2 integrins due to inhibition of PKA is abolished by pharmacologic MLCK inhibition. Inhibition of MLCK also blocked β2 integrin-dependent neutrophil adhesion achieved by inhibition of PKA, as well as neutrophil migration along towards a PKA inhibitor. These findings demonstrate that PKA regulation of β2 integrin affinity activation and subsequent neutrophil activation is via an MLCK-dependent pathway.
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    Redox Thermodynamics of Dehaloperoxidase-Hemoglobin
    (2010-04-09) D'Antonio, Edward Lawrence; Dr. Robert B. Rose, Committee Member; Dr. Edmond F. Bowden, Committee Chair; Dr. Stefan Franzen, Committee Member; Dr. David C. Muddiman, Committee Member
    Dehaloperoxidase-hemoglobin (DHP) is a small intracellular hemoglobin found in the terebellid polychaete Amphitrite ornata. This heme protein can transport oxygen to the cells, but it also has moderate peroxidase activity. As a result of its hybrid functionality between the globin and peroxidase classes of heme proteins, various properties of DHP have been found to be unique. Among one of these properties is the Fe(III)/Fe(II) formal reduction potential, which has been determined herein, in solution and surface-bound. Furthermore, electrochemical investigations of DHP have not been explored to any significant extent. The formal reduction potential of DHP is much more positive than any known peroxidase and more positive than any intracellular globin. A thermodynamic analysis of the free energy contributions that give rise to this high reduction potential is due to the redox-coupled conformational change that happens with the distal histidine (H55) between Fe(III) and Fe(II) oxidation states. DHP is also known to bind inhibitors, such as para-halophenols inside its distal binding pocket and it can bind substrates, such as 2,4,6-trihalophenols on the external side of the protein. When DHP is exposed to these halophenols, it was determined that a modulation in the Fe(III)/Fe(II) oxidation/reduction potential occurs and the result is more substantial for internal binding than external. Both binding modes cause the shift in potential to be negative. Proximal region mutations were also explored for the purpose of installing in the socalled Asp-His-Fe triad into DHP, which is generally found in peroxidases but not globins, so that the “push†effect could be studied. The “push†effect refers to there being anionic character on the proximal ligand of a heme peroxidase, which has a role in “pushing†apart the O-O bond of hydrogen peroxide in the peroxidase reaction. So far a globin model system has not been made successful. These results show that by making this type of mutation into DHP (i.e. the M86D mutation), the mutant causes H55 to coordinate as the sixth ligand to the iron atom and inhibits all peroxidase activity, under physiological conditions. The study clarifies that globins simply do not have this structural feature because they are not designed to carry out peroxidase chemistry. Electrochemical results aided in characterizing if these mutants had established the triad. Other structural techniques employed were 13C-NMR, Xray crystallography, and resonance Raman spectroscopy. Finally, an electrochemical study of the Fe(III)/Fe(II) redox couple of DHP adsorbed to a self-assembled monolayer surface on a gold working electrode was carried out for method development purposes. By establishing the optimum conditions in obtaining reversible cyclic voltammetry while maintaining surface stability of DHP, this groundwork will be useful for future studies directed at immobilized DHP electrocatalysis.