Browsing by Subject "Zinc"
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Item Applications of bis(imino)acenaphthene and investigation of boron arsenide as a high thermal conductivity material(2015-05) Evans, Daniel Anthony; Cowley, Alan H.; Jones, Richard A; Humphrey, Simon M; Anslyn, Eric V; Ekerdt, John GAbstract: Functionalization of the ubiquitous bis(imino)acenaphthene ligand class has been explored. The successful functionalization of this ligand type was found to be dependent upon the steric congestion encompassing the N-C-C-N fragment of the aryl substituted BIAN ligand. The sterically directed functionalization was found to proceed via either a radical backbone dearomatization route or a nucleophilic imine C-alkylation pathway. The structures of each of the functionalized BIAN derivatives were examined by means of single crystal X-ray crystallography. The foregoing reactions were also probed by EPR spectroscopy and DFT-D calculations in order to help elucidate the nature of the driving forces that are involved in BIAN functionalization. A series of aryl substituted BIAN zinc(II) chloride complexes were also prepared and their photophysical properties were investigated. Initially, four different methylated aryl substituents were examined, namely the 4-methylphenyl, 3,5-dimethylphenyl, 2,4,6-trimethylphenyl, and 2-methylphenyl derivatives. Examination of these four complexes revealed them to be non-emissive in solution. However, it was also determined that the 4-methylphenyl and 3,5-dimethylphenyl substituted complexes were emissive in the solid state. On the other hand, the 2,4,6-trimethylphenyl, and 2-methylphenyl complexes were found to be non-emissive in the solid state. The origins of the emissions of the foregoing complexes were also probed by means of TD-DFT calculations. The tuning of the stereoelectronic properties of a series of para-substituted aryl BIAN zinc(II) chloride complexes was undertaken with the view to modifying their solid state photophysical properties. For example, changing the electronic properties of the flanking para-substituted aryl substituents permitted tunability within the range of the red-orange-yellow emissions. Tunability was also achieved by employing a variety of different recrystallization techniques for growing the various structures, polymorphs, and solvatomorphs of each BIAN zinc(II) chloride complex. Boron arsenide, a somewhat neglected semiconductor compound, has been examined for its potential use as a high thermal conductivity material. High quality single crystal BAs microstructures have been synthesized and characterized by means of powder X-ray diffraction, X-ray photoelectron spectroscopy, Raman spectroscopy, and scanning electron microscopy. The thermal conductivity properties of the BAs microstructures have been probed using microheater devices.Item Characterization of the interactions of zinc with known and novel allosteric modulators of glycine receptor function(2016-12) Cornelison, Garrett Lee; Mihic, S. John; Aldrich, Richard W; Harris, R. Adron; Stavchansky, Salomon AThe glycine receptor is a member of the Cys-loop receptor superfamily of ligand-gated ion channels and is implicated as a possible therapeutic target for the treatment of diseases such as alcoholism and inflammatory pain. In humans, four glycine receptor subtypes (α1, α2, α3, and β) co-assemble to form pentameric channel proteins as either α homomers or αβ heteromers. To date, few agents have been identified that can selectively modulate the glycine receptor, especially those possessing subtype specificity. We used a cell-based method of phage display panning, coupled with two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, to identify novel heptapeptide modulators of the α1β glycine receptor. Peptides were identified that act with selectivity on α1β and α3β compared to α2β glycine receptors. In addition, peptide activity at the glycine receptor decreased when zinc was chelated by tricine, similar to previous observations of a decrease in ethanol’s enhancing actions at the receptor in the absence of zinc. Zinc is an allosteric modulator of glycine receptor function, enhancing the effects of glycine at nanomolar to low micromolar concentrations, and inhibiting its effects at higher concentrations. As zinc is present physiologically at various concentrations within this range, it is capable of influencing glycine receptor function, including modulation by other pharmacological agents; however, the magnitude of this effect and its possible relevance are not known. I therefore investigated the utility of previously-described “zinc-enhancement insensitive” α1 glycine receptor mutants D80A, D80G, and W170S to probe for interactions between zinc and other allosteric modulators at the glycine receptor. Interestingly, I found that only the W170S mutation conferred complete abolishment of zinc enhancement across a variety of agonist and zinc concentrations. Using α1 W170S receptors, I established that in addition to ethanol, zinc also interacts with inhaled drugs of abuse, but not volatile anesthetics, to synergistically enhance channel function. Additionally, I determined that this interaction is abolished at higher zinc concentrations, when receptor-enhancing bindings sites are saturated, suggesting a mechanism by which modulators such as ethanol and inhalants are capable of increasing receptor affinity for zinc in addition to enhancing channel function on their own.Item A critical role for zinc in ethanol action at the glycine receptor(2012-05) McCracken, Lindsay Marie; Harris, R. Adron; Gore, Andrea; Duvauchelle, Christine; Morrisett, Richard; Trudell, JamesEthanol is a widely used drug, yet an understanding of its sites and mechanisms of action remains incomplete. Among the protein targets of ethanol are glycine receptors (GlyRs). In addition to ethanol, zinc also modulates GlyR function. Although the individual effects of zinc and alcohols on GlyR function have been well studied, the combined effects of these agents have not been thoroughly examined. This project investigated the effects of zinc on alcohol action at the glycine receptor (GlyR). In Aim 1, the effects of zinc on ethanol modulation of GlyR function were tested and characterized in three GlyR [alpha] subunits ([alpha]1-3). Aim 2 explored a site of action for the augmenting effects of zinc on ethanol action at the GlyR. Mutant D80A GlyRs, which lack a zinc binding site (D80), were constructed and allowed us determine if this zinc binding site is important for the zinc/ethanol interactions that were observed in Aim 1. The effects of ethanol were reduced in mutant D80A GlyRs compared to wild type (WT). In addition, manipulating zinc levels in our buffers either by adding or chelating zinc did not change the magnitude of ethanol enhancement of mutant D80A GlyRs as it did in WT GlyRs suggesting that the D80 position is important for zinc modulation of ethanol action. Finally, Aim 3 extended the findings from Aims 1 and 2 by evaluating the effects of a GlyR point-mutation on alcohol consumption and other behavioral tests in mice. Glra1(D80A) knock-in mice provided an animal model for behavioral studies of zinc/ethanol interactions at the GlyR and showed decreased alcohol consumption and preference compared to their WT littermates. In addition, D80A KI mice had increased startle responses compared to their WT littermates. Other behavioral tests were also conducted including tests of ethanol motor incoordination and strychnine induced convulsions; there were no differences detected between KI and WT mice in these assays. Overall, our findings demonstrate that zinc is critical in determining the effects of ethanol at GlyRs and suggest that zinc signaling at the D80 position may be important for mediating the behavioral effects of ethanol action at GlyRs.Item Dependence of allosteric modulation of glycine receptor function on agonist efficacy(2017-08-11) Farley, Nicole-Marie Margot Hetzer; Mihic, S. John; Harris, R. Adron; Aldrich, Richard W; Pierce-Shimomura, Jon T; Wilcox, Richard EThe glycine receptor (GlyR) is a ligand-gated chloride channel that mediates inhibitory neurotransmission in the brain and spinal cord. Numerous allosteric modulators act on the GlyR including divalent cations, such as zinc, as well as drugs of abuse including ethanol, inhalants, anesthetics and cannabinoids. GlyRs mediate some of the rewarding effects of addictive drugs and modulate drug related behaviors through activity in the mesolimbic dopamine reward pathway. GlyR activity, however, can differ depending on whether the receptor is activated by the high-efficacy agonist, glycine, or taurine which has much lower efficacy at wild-type GlyRs. As glycine and taurine are believed to activate the GlyR in vivo, it is crucial that we understand receptor function and allosteric modulation of receptors in response to both agonists. We used two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes to study the effects of zinc and ethanol on wildtype glycine receptors activated by glycine or taurine. We determined that the magnitude of allosteric modulation was higher, overall, at taurine-gated receptors and hypothesized that this may be related to the difference in efficacies. Considering that GlyR mutants can affect agonist sensitivity and the response to allosteric modulators, we wondered whether changes in allosteric modulation at mutant receptors could be due to changes in agonist efficacy caused by these mutations. We tested this hypothesis by characterizing ethanol and zinc modulation of taurine currents at GlyR mutants that showed an increase ([alpha]1[superscript W170S]) or decrease ([alpha]1[superscript A52S]) in taurine efficacy. We found that the W170S mutation increases the relative efficacy of taurine to a level that is comparable with glycine and abolishes ethanol enhancement of maximally-effective taurine currents. We determined that the difference in ethanol potentiation of low taurine currents between W170S and WT receptors is due to zinc enhancement of WT currents. Ethanol modulation of these receptors was equal in the presence of tricine. This suggests that ethanol also increases taurine affinity at W170S. A52S, on the other hand, displayed reduced taurine efficacy and increased ethanol modulation. This work provides evidence of a mechanism by which the degree of allosteric modulation of glycine receptor function is dependent on agonist efficacy.Item A protocol to evaluate the adsorptive removal of dissolved copper and zinc from highway runoff(2014-05) Ernst, Clayton Owen; Katz, Lynn EllenThe increasing urbanization of landscapes significantly alters the surface water hydrology of impacted watersheds. As a side effect, stormwater discharges to receiving water bodies are often of decreased quality due to pollutants deposited on impervious urban surfaces being entrained by runoff. A pertinent example of this problem is the presence of copper and zinc in highway runoff. Both copper and zinc have been shown to exert toxic effects on aquatic micro- and macro-biota. Copper in particular has been shown to harmfully disrupt the olfactory nervous system of fish species at concentrations as low as 3 [mu]g/L. To meet these limits, treatment of highway runoff for the removal of copper and zinc is necessary. However, due to the complexities associated with the behavior of heavy metals in natural systems, the appropriateness of removal techniques will necessarily depend on a variety of system-specific factors and chemical characteristics of highway runoff. Adsorption has been shown to be generally effective in the removal of dissolved heavy metals, but the choice of adsorptive media is again dependent on system-specific parameters. This study developed and evaluated a column testing protocol that can be used to quickly and reliably evaluate adsorptive removal of dissolved heavy metals from highway runoff. The protocol is demonstrated in an evaluation of iron oxide, manganese oxide, crab shell, concrete, and bone meal media for removing dissolved copper and zinc from highway runoff. The performance of these media was assessed as a function of various runoff characteristics including pH, ionic strength, alkalinity, and total organic carbon. The methodology was used to show that iron oxide media in combination with crab shell or concrete media provided the most effective removal of copper and zinc from highway runoff. Through this study, the convenience, flexibility, and robustness of the proposed protocol are compellingly established.Item The relationship between glycine receptor agonist efficacy and allosteric modulation(2014-05) Kirson, Dean; Mihic, S. JohnThe glycine receptor (GlyR) is a ligand-gated ion channel member of the cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Both glycine and the partial agonist taurine act as endogenous ligands of the GlyR. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse, such as ethanol. As a partial agonist, taurine has a decreased efficacy relative to glycine, resulting in a decreased maximum response. We investigated the effects of ethanol, anesthetics, inhalants, and zinc to determine if these allosteric modulators could increase the efficacy of the taurine-activated GlyR. Whole cell recordings of wild type GlyR revealed that each of the allosteric modulators potentiated currents generated by saturating concentrations of taurine but not glycine, implying an increase in efficacy. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of these allosteric modulators with zinc to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by another allosteric modulator, indicating no synergistic effects on efficacy. We next investigated the actions of ethanol and isoflurane on the taurine-activated GlyR at the single channel level, finding that both allosteric modulators stabilized the channel open state, increasing the efficacy of the taurine-activated GlyR. We previously identified a mutation in the ligand-binding domain of the GlyR (D97R) that produces spontaneously activating channels, on which taurine has increased efficacy. We identified a residue, R131, as a possible binding partner of D97 in forming an electrostatic interaction that holds the channel in the closed state. We found that disruption of this interaction results in greatly increased taurine efficacy, indicating that efficacy for partial agonists may be determined by agonist ability to break this bond early in the activation process following binding. Thus we find differential mechanisms of allosteric modulation and efficacy determinations for the GlyR when activated by taurine vs. glycine.Item Structural analysis and discovery of lead compounds for the fungal methionine synthase enzyme(2013-12) Ubhi, Devinder Kaur; Robertus, Jon D.Methionine synthases catalyze methyl transfer from 5-methyl-tetrahydrofolate (5-methyl-THF) to L-homocysteine (Hcy) in order to generate methionine (Met). Mammals, including humans, use a cobalamin dependent form, while fungi use a cobalamin independent protein called Met6p. The large structural differences between them make Met6p a potential anti-fungal drug target. Met6p is a 90 kDa protein with the active site located between two (βα)₈ barrels. The active site has a catalytic Zn²+ and binding sites for the two substrates, Hcy and folate. I present the crystal structures of three engineered variants of the Met6p enzyme from Candida albicans. I also solved Met6p in complex with several substrate and product analogs, including Hcy, Met, Gln, 5-methyl-THF-Glu₃ and Methotrexate-Glu₃ (MTX-Glu₃), and the bi-dentate ligand S-adenosyl homocysteine. Also described is a new fluorescence-based activity assay monitoring Hcy. Lastly, a high-throughput Differential Scanning Fluorimetry (DSF) assay was used to screen thousands of compounds in order to identify ligands which bind Met6p. My work details the mode of interaction of Hcy and folate with the Met6p protein. Several residues important to activity were discovered, like Asn 126 and Tyr 660, and proven to be important by site directed mutagenesis. Structural analysis revealed an important aspect of the mechanism. When Hcy binds to its pocket it makes strong ion pairs with the enzyme. In particular, 614 moves toward the substrate amine and triggers a rearrangement of active site loops; this draws the catalytic Zn²+ toward the Hcy thiol where a new ligand bond is formed, activating the thiol for methyl transfer. The work presented here lays the groundwork for structure based drug design and makes the development of Met6p specific bi-dentate ligands feasible. The fluorescence based activity assay I developed was successfully used to test the folate analog MTX-Glu₃, which inhibits with an IC₅₀ of ~4 mM. I also discovered our first bi-dentate ligand in the form of S-adenosyl homocysteine.Item Zinc interactions with allosteric modulators at the glycine receptor(2014-08) Cornelison, Garrett Lee; Mihic, S. JohnThe glycine receptor (GlyR) is a ligand-gated ion channel member of the Cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Zinc is a potent allosteric modulator of GlyR function, enhancing GlyR activity at low nM to 10[mu]M concentrations while inhibiting GlyR activity at higher concentrations. We investigated sources of contaminating zinc, identifying low nM levels of zinc in ultrapure H₂O, powdered reagents used in the preparation of common electrophysiological buffers, and in polystyrene pipets. These low levels of zinc were capable of enhancing GlyR function. These findings suggest that without checking for this effect using a zinc-chelator such as tricine, one cannot assume that responses elicited by glycine applied alone are not necessarily also partially due to some level of allosteric modulation by zinc. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of zinc with drugs of abuse on taurine-activated GlyR to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by drugs of abuse, indicating no synergistic effects on efficacy when receptors are saturated with taurine as may be seen during synaptic events in vivo. Finally, we utilized phage display to identify novel peptide modulators of the GlyR. We tested 26 peptides against [alpha1beta] GlyRs, identifying peptides with various levels of activity on GlyR function. We demonstrated that these modulators were zinc-dependent, as their effects on GlyR activity were abolished in the presence of the zinc-chelating agent tricine. Together, these data indicate the importance of accounting for the effects of zinc when studying the function of the GlyR, as even low levels of zinc that can be found as contaminants in labware and buffers can affect GlyR function and responses to various allosteric modulators, including drugs of abuse.