Inflammatory and neuropathic pains arise in part from sensitization at nociceptive synapses in the spinal cord. Activity-dependent signaling cascades converge onto the tyrosine kinase Src, which participates in augmenting the function of N-methyl-D-aspartate receptors (NMDARs) and thus potentiates the nociceptive system. Src is capable of these effects because it is anchored to the NMDAR complex via an adaptor protein called NADH dehydrogenase subunit 2 (ND2). There is evidence that this interaction occurs between amino acids 40-49 of Src and amino acids 310-321 of ND2. I have determined that a peptide consisting of amino acids 310-321 of ND2, and affixed to the HIV Tat domain for cell permeability, is capable of alleviating tactile allodynia induced by Complete Freund's Adjuvant (CFA) in rats. Src40-49Tat was not effective in two models of inflammatory pain. This work further implicates the Src-ND2 interaction in pain hypersensitivity and suggests that Tat ND2 310-321 may alleviate it.

Inflammatory and neuropathic pains arise in part from sensitization at nociceptive synapses in the spinal cord. Activity-dependent signaling cascades converge onto the tyrosine kinase Src, which participates in augmenting the function of N-methyl-D-aspartate receptors (NMDARs) and thus potentiates the nociceptive system. Src is capable of these effects because it is anchored to the NMDAR complex via an adaptor protein called NADH dehydrogenase subunit 2 (ND2). There is evidence that this interaction occurs between amino acids 40-49 of Src and amino acids 310-321 of ND2. I have determined that a peptide consisting of amino acids 310-321 of ND2, and affixed to the HIV Tat domain for cell permeability, is capable of alleviating tactile allodynia induced by Complete Freund's Adjuvant (CFA) in rats. Src40-49Tat was not effective in two models of inflammatory pain. This work further implicates the Src-ND2 interaction in pain hypersensitivity and suggests that Tat ND2 310-321 may alleviate it.

Hemoglobin plays an important role in transporting oxygen in human beings and other mammals. Hemoglobin is a tetrameric protein composed of two alpha and two beta subunits. The £\ and £] subunits are both necessary and the stoichiometric ratio of the two dislike subunits is critical for hemoglobin to perform its oxygen-carrying function properly. To better understand the coupling between the £\ and £] subunits and the subunit disassembly pathway, p-hydroxymercuri-benzoate (PMB) has been used to react with the cysteine residues in hemoglobin. The hemoglobin tetramer becomes unstable and disassembles into £\ and £] subunits when the cysteine sites are perturbed upon reacting with PMB. There are three kinds of cysteine residues, £]93, £\104 and £]112, in human hemoglobin. The reactivity of different cysteine residues with PMB and their reaction sequence have been studied via the Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The resonance Raman spectroscopy has been used to investigate the structural changes of hemoglobin accompanying the PMB-modification under the oxygenated and deoxygenated conditions. At last, a hemoglobin subunit disassembly mechanism is proposed and the site-specific roles of cysteine residues in human hemoglobin are discussed in detail.

Calpains are calcium-dependent, intracellular, multi-domain cysteine proteases involved in many physiological functions regulated by calcium signaling, including cell motility. How calpains are activated in the cell is still unknown because the resting intracellular concentration of Ca2+ is orders of magnitude lower than that needed for half-maximal activation of the enzyme in vitro. Several stratagems by which calpains might overcome this Ca2+ concentration differential have been proposed. It is possible that post-translational modifications like phosphorylation, or accessory proteins that bind to calpain, might facilitate the enzyme’s activation at lower than optimal Ca2+ concentrations. Autoproteolysis (autolysis) and subunit dissociation are two other proposed activation mechanisms that could release constraints on the calpain core by breaking the link between the anchor helix and the small subunit to allow the active site to form. By measuring the rate of autolysis at different sites in calpain, it was demonstrated that while the anchor helix is one of the first targets to be cut, several other potentially inactivating autolysis sites, particularly in Domain III, can also be cleaved within the first minute. Thus autolytic activation would go hand in hand with inactivation. By fractionating and identifying calpain 2 autolysis fragments, I show that the small subunit does not dissociate away from the large subunit, but is proteolyzed to a 40-45 k heterodimer of the penta-EF-hand Domains IV and VI. It is likely that this autolysis-generated heterodimer has previously been misidentified as the small subunit domain VI homodimer that would be produced by subunit dissociation. A calpastatin affinity column was constructed and used to capture recombinant calpain 2 from bacterial cell lysate. This affinity column provides a tool to screen for and capture calpain complexed to potential binding partners in the presence of Ca2+. Here I propose a model for calpain 2 activation in vitro that does not involve autolysis, subunit dissociation, or calpain activators. / Thesis (Master, Biochemistry) -- Queen's University, 2010-10-25 16:03:52.364

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Thesis (Ph.D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Biochemistry. Title from title-page of electronic thesis. Bibliography: leaves 92 - 100.

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The development of microbial resistance to practically all currently used antimicrobial agents has spurred efforts to develop new antibiotics and to identify novel targets in bacterial cells. This review summarizes the evidence for inhibition of bacterial ribosomal subunit formation as a target for many antibiotics distinct from their well-known inhibition of translation. Features of a model to explain this activity are explored. Results are presented to show the accumulation of both 30S and 50S ribosomal subunit precursors in antibiotic inhibited cells. These precursors have been characterized and are shown to bind radio-labeled drugs. Pulse and chase labeling studies have revealed the slower rates of subunit synthesis in drug treated cells compared with uninhibited controls. Resynthesis of subunits after antibiotic removal precedes recovery of control protein synthesis capacity, consistent with the model presented. Also certain mutant strains defective in different ribonuclease activities are more susceptible to antibiotic inhibition of assembly as predicted. Results indicating the equivalence of assembly inhibition and translational inhibition are described. Lastly, the identification of a 50S subunit precursor particle as a substrate for rRNA methyltransferase activity is shown. The weight of evidence presented clearly indicates that ribosomal antibiotics have a second target in cells. Inhibition of cell growth and subsequent cell death results from the activity of these antibiotics on the combined targets. The possibility of designing assembly specific inhibitors is discussed.

Bacteria are the most abundant organisms on the planet. They live almost in all environments, including those that are most extreme. All land and water ecosystems depend heavily upon their activity. Bacteria play essential roles in cycling of nutrients such as carbon, nitrogen, and sulphur. Due to their short cell cycle, they must be able to swiftly adapt to the conditions of their habitat to survive. Microbial growth itself is an autocatalytic process. There are three distinct phases of the growth curve: lag, exponential (log), and stationary. Bacterial cells must change their gene expression between these phases in order to adapt to the new conditions. The first stage of gene expression is transcription. The key enzyme of this stage is RNA polymerase (RNAP) that transcribes DNA into RNA. RNAP is regulated by a number of accessory proteins and also small molecule effectors. Understanding how RNAP functions is essential for understanding how bacteria cope with changing environments. This Thesis presents studies of selected aspects of bacterial gene expression regulation at the level of transcription, using Bacillus subtilis as the model organism. The first part of this Thesis focuses on protein determinants of the ability of RNAP to be regulated by the concentration of the initiating nucleoside...