Research and Publications - Department of Chemistry & Biochemistry
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Item On Osmious Acid, and the Position of Osmium in the List of Elements(American Journal of Science, 1860) Mallet, John WilliamsItem La Fayette Guild(Paul B. Hoeber, 1935-03) Carmichael, Emmett B.Item Charles Alexander Pope(P.B. Hoeber, 1940-09-01) Carmichael, Emmett B.Item William Owen Baldwin(Paul B. Hoeber, 1942) Carmichael, Emmett B.Item Constituents of the Blood of the Hibernating and Normal Rattlesnake, Crotalus Horridus(1945-12-01) Carmichael, Emmett B.; Petcher, Paul W.Item Tryptophan operon read-through. Isolation and characterization of an abnormally long tryptophan synthetase alpha subunit from a frame-shift mutant of Escherichia coli(American Society for Biochemistry and Molecular Biology, 1975-06-25) Hardman, John K.; Berger, Hillard; Goodman, MyronA new mutant strain of Escherichia coli, strain ICR-47, contains a frame-shift mutation in the trpA gene, the gene most distal to the operator in the trp operon. Mapping experiments indicate that the lesion is located at a site within 10 to 15% of the end of this gene. The mutation results in "out-of-phase" translation of the distal portion of the trp mRNA; normal translational termination signal(s) are not encountered and a trpA gene product longer than the wild type protein is produced. As with the other enzymes produced from this operon, the in vivo level of the altered protein (the alpha subunit of the tryptophan synthetase enzyme complex) is controlled by exogenous L-tryptophan. The altered alpha subunit from the strain ICR-47 has been isolated and characterized. Molecular weight estimations indicate a molecular weight of approximately 37,000, an increase beyond the wild type enzyme corresponding to an additional 50 to 70 amino acid residues. The protein has a new COOH-terminal amino acid sequence. Results of preliminary hybridization experiments suggest that the ICR-47 mRNA, which is necessarily longer than that needed to code for wild type enzyme, is not detectably different in size from wild type mRNA. The enzymatic properties of the ICR-47 alpha subunit indicates a greatly reduced ability of the mutant subunit to combine functionally with wild type beta2 subunit, the second protein component in the tryptophan synthetase enzyme complex. In contrast, only 40 to 50% of the intrinsic enzymatic activity of the alpha subunit is lost.Item Structural Characterization of Nitrimyoglobin(American Society for Biochemistry and Molecular Biology, 1989-04-15) Bondoc, Laureano L.; Timkovich, RussellNitrimyoglobin was formed in greater than 94% yield by a simple reaction between excess nitrite and horse heart metmyoglobin at pH 5.5. This dark green pigment was shown by 1H NMR spectroscopy to be a single, pure product with a well defined tertiary structure that is highly similar to the starting myoglobin. Electronic spin states parallel those of myoglobin, although the relaxation times differ. Ligand binding reactions of nitrimyoglobin parallel those of normal myoglobin, but lead to a unique series of UV-visible spectra. In the ferrous state, nitrimyoglobin reversibly binds O2 with half-saturation of sites at an O2 partial pressure of 10.4 ± 1.4 mm Hg. 1H NMR data indicate that the altered heme of nitrimyoglobin has not undergone reaction at any meso proton position, nor has it been partially saturated to the level of a chlorin. 15N NMR spectra indicate that only a single nitrogen was added to the protein as a nitro group. Extraction of the modified heme from nitrimyoglobin and spectroscopic characterization of the nitriheme by infrared spectroscopy and of the free base porphyrin methyl ester derived from nitriheme by 1H NMR indicate that the modification is regiospecific. The heme in nitrimyoglobin is 3-(trans-2-nitrovinyl)-2,7,12,18-tetramethyl-8-vinylporphyrin-13,17-dipropionic acid. In the Fisher nomenclature scheme, the 2-vinyl substituent is the site of modification and has been converted to a nitrovinyl group by substitution of a proton by -NO2.Item Radical reactions involving cyclodextrins and model sugars(UMI Company, 1997) Lehmann, Marc N.Time-resolved EPR/laser flash photolysis studies of aqueous solutions of D-glucose, alpha-methyl-D-glucose, maltose and alpha-, beta- and gamma-cyclodextrin (CD) with acetone, pyruvic acid (PA), methylethylketone (MEK) and levulinic acid (LA) showed that the excited triplet state of these ketones could be used to selectively produce different polarized sugar radicals depending on the substrate used. For both D-glucose and maltose the C1 carbon centered radical was observed to be most intense and thought to be a consequence of the anomeric effect which favors binding of the ketone to the hydroxyl group situated on this carbon. For alpha-methyl-D-glucose the C7, C6 and C2 centered radicals were most intense and their selective formation is attributed to affinity effects. The following CD radicals were identified. From alpha-CD; C3 and C5 centered radicals. From beta-CD; C3, C5 and C1. From gamma-CD; C1 and C6. A C5 carbon centered radical formed from glucopyranoside ring-opening of the C1 radical was also observed for beta-CD and gamma-CD. Changes in the intensity of the spectra of the radicals observed between alpha-, beta- and gamma-CD were explained to arise from the extent ketone binding to the cyclodextrin cavity.Item Electron Flow between Photosystem II and Oxygen in Chloroplasts of Photosystem I-deficient Algae Is Mediated by a Quinol Oxidase Involved in Chlororespiration(American Society for Biochemistry and Molecular Biology, 2000-06-09) Cournac, Laurent; Redding, Kevin; Ravenel, Jacques; Rumeau, Dominique; Josse, Eve-Marie; Kuntz, Marcel; Peltier, GillesIn Chlamydomonas reinhardtii mutants deficient in photosystem I because of inactivation of the chloroplast genes psaA or psaB, oxygen evolution from photosystem II occurs at significant rates and is coupled to a stimulation of oxygen uptake. Both activities can be simultaneously monitored by continuous mass spectrometry in the presence of18O2. The light-driven O2 exchange was shown to involve the plastoquinone pool as an electron carrier, but not cytochrome b 6 f. Photosystem II-dependent O2 production and O2 uptake were observed in isolated chloroplast fractions. Photosystem II-dependent oxygen exchange was insensitive to a variety of inhibitors (azide, carbon monoxide, cyanide, antimycin A, and salicylhydroxamic acid) and radical scavengers. It was, however, sensitive to propyl gallate. From inhibitors effects and electronic requirements of the O2 uptake process, we conclude that an oxidase catalyzing oxidation of plastoquinol and reduction of oxygen to water is present in thylakoid membranes. From the sensitivity of flash-induced O2 exchange to propyl gallate, we conclude that this oxidase is involved in chlororespiration. Clues to the identity of the protein implied in this process are given by pharmacological and immunological similarities with a protein (IMMUTANS) identified in Arabidopsis chloroplasts.Item Evidence for two active branches for electron transfer in photosystem I(The National Academy of Sciences, 2001) Guergova-Kuras, Mariana; Boudreaux, Brent; Joliot, Anne; Joliot, Pierre; Redding, KevinAll photosynthetic reaction centers share a common structural theme. Two related, integral membrane polypeptides sequester electron transfer cofactors into two quasi-symmetrical branches, each of which incorporates a quinone. In type II reaction centers [photosystem (PS) II and proteobacterial reaction centers], electron transfer proceeds down only one of the branches, and the mobile quinone on the other branch is used as a terminal acceptor. PS I uses iron-sulfur clusters as terminal acceptors, and the quinone serves only as an intermediary in electron transfer. Much effort has been devoted to understanding the unidirectionality of electron transport in type II reaction centers, and it was widely thought that PS I would share this feature. We have tested this idea by examining in vivo kinetics of electron transfer from the quinone in mutant PS I reaction centers. This transfer is associated with two kinetic components, and we show that mutation of a residue near the quinone in one branch specifically affects the faster component, while the corresponding mutation in the other branch specifically affects the slower component. We conclude that both electron transfer branches in PS I are active.Item Theory of electrical rectification in a molecular monolayer(American Physical Society, 2001) Krzeminski, C.; Delerue, C.; Allan, G.; Vuillaume, D.; Metzger, R.M.The current-voltage characteristics in Langmuir-Blodgett monolayers of γ-hexadecylquinolinium tricyanoquinodimethanide (C16H33Q-3CNQ) sandwiched between Al or Au electrodes is calculated, combining ab initio and self-consistent tight binding techniques. The rectification current depends on the position of the LUMO and HOMO relative to the Fermi levels of the electrodes as in the Aviram-Ratner mechanism, but also on the profile of the electrostatic potential which is extremely sensitive to where the electroactive part of the molecule lies in the monolayer. This second effect can produce rectification in the direction opposite to the Aviram-Ratner prediction.Item Synthesis and Characterization of Multifunctional Chitosan- MnFe2O4 Nanoparticles for Magnetic Hyperthermia and Drug Delivery(MDPI, 2010) Kim, Dong-Hyun; Nikles, David E.; Brazel, Christopher S.; University of Alabama TuscaloosaMultifunctional nanoparticles composed of MnFe2O4 were encapsulated in chitosan for investigation of system to combine magnetically-triggered drug delivery and localized hyperthermia for cancer treatment with the previously published capacity of MnFe2O4 to be used as an efficient MRI contrast agent for cancer diagnosis. This paper focuses on the synthesis and characterization of magnetic MnFe2O4 nanoparticles, their dispersion in water and their incorporation in chitosan, which serves as a drug carrier. The surface of the MnFe2O4 nanoparticles was modified with meso-2,3-di-mercaptosuccinic acid (DMSA) to develop stable aqueous dispersions. The nanoparticles were coated with chitosan, and the magnetic properties, heat generation and hydrodynamic size of chitosan-coated MnFe2O4 were evaluated for various linker concentrations and in a range of pH conditions.Item Catecholamines up integrates dopamine synthesis and synaptic trafficking(Wiley, 2011) Wang, Zhe; Ferdousy, Faiza; Lawal, Hakeem; Huang, Zhinong; Daigle, J. Gavin; Izevbaye, Iyare; Doherty, Olugbenga; Thomas, Jerrad; Stathakis, Dean G.; O'Donnell, Janis M.; University of Alabama Tuscaloosa; University of Mississippi Medical Center; University of Mississippi; University of VirginiaThe highly reactive nature of dopamine renders dopaminergic neurons vulnerable to oxidative damage. We recently demonstrated that loss-of-function mutations in the Drosophila gene Catecholamines up (Catsup) elevate dopamine pools but, paradoxically, also confer resistance to paraquat, an herbicide that induces oxidative stress-mediated toxicity in dopaminergic neurons. We now report a novel association of the membrane protein, Catsup, with GTP cyclohydrolase rate-limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis and tyrosine hydroxylase, rate-limiting enzyme for dopamine biosynthesis, which requires BH4 as a cofactor. Loss-of-function Catsup mutations cause dominant hyperactivation of both enzymes. Elevated dopamine levels in Catsup mutants coincide with several distinct characteristics, including hypermobility, minimal basal levels of 3,4-dihydroxy-phenylacetic acid, an oxidative metabolite of dopamine, and resistance to the vesicular monoamine transporter inhibitor, reserpine, suggesting that excess dopamine is synaptically active and that Catsup functions in the regulation of synaptic vesicle loading and release of dopamine. We conclude that Catsup regulates and links the dopamine synthesis and transport networks.Item Inhibitors of LRRK2 kinase attenuate neurodegeneration and Parkinson-like phenotypes in Caenorhabditis elegans and Drosophila Parkinson's disease models(Oxford University Press, 2011) Liu, Zhaohui; Hamamichi, Shusei; Lee, Byoung Dae; Yang, Dejun; Ray, Arpita; Caldwell, Guy A.; Caldwell, Kim A.; Dawson, Ted M.; Smith, Wanli W.; Dawson, Valina L.; University of Alabama Tuscaloosa; University of Maryland Baltimore; Johns Hopkins University; University of Alabama BirminghamMutations in leucine-rich repeat kinase 2 (LRRK2) have been identified as a genetic cause of familial Parkinson's disease (PD) and have also been found in the more common sporadic form of PD, thus positioning LRRK2 as important in the pathogenesis of PD. Biochemical studies of the disease-causing mutants of LRRK2 implicates an enhancement of kinase activity as the basis of neuronal toxicity and thus possibly the pathogenesis of PD due to LRRK2 mutations. Previously, a chemical library screen identified inhibitors of LRRK2 kinase activity. Here, two of these inhibitors, GW5074 and sorafenib, are shown to protect against G2019S LRRK2-induced neurodegeneration in vivo in Caenorhabditis elegans and in Drosophila. These findings indicate that increased kinase activity of LRRK2 is neurotoxic and that inhibition of LRRK2 activity can have a disease-modifying effect. This suggests that inhibition of LRRK2 holds promise as a treatment for PD.Item Polymer Micelles with Crystalline Cores for Thermally Triggered Release(American Chemical Society, 2012) Glover, Amanda L.; Nikles, Sarah M.; Nikles, Jacqueline A.; Brazel, Christopher S.; Nikles, David E.; University of Alabama Tuscaloosa; University of Alabama BirminghamInterest in the use of poly(ethylene glycol)-b-polycaprolactone diblock copolymers in a targeted, magnetically triggered drug delivery system has led to this study of the phase behavior of the polycaprolactone core. Four different diblock copolymers were prepared by the ring-opening polymerization of caprolactone from the alcohol terminus of poly(ethylene glycol) monomethylether, M-n approximate to 2000. The critical micelle concentration depended on the degree of polymerization for the polycaprolactone block and was in the range of 2.9 to 41 mg/L. Differential scanning calorimetry curves for polymer solutions with a-concentration above the critical micelle concentration showed a melting endotherm in the range of 40 to 45 degrees C, indicating the polycaprolactone core was semicrystalline. Pyrene was entrapped in the micelle core without interfering with the ability of the polycaprolactone to crystallize. When the polymer solution was heated above the melting point of the micelle core, the pyrene was free to leave the core. Temperature-dependent measurements of the critical micelle concentration and temperature-dependent dynamic light scattering showed that the micelles remain intact at temperatures above the melting point of the polycaprolactone core.Item Nrf2b, Novel Zebrafish Paralog of Oxidant-responsive Transcription Factor NF-E2-related Factor 2 (NRF2)(American Society of Biochemistry and Molecular Biology, 2012) Timme-Laragy, Alicia R.; Karchner, Sibel I.; Franks, Diana G.; Jenny, Matthew J.; Harbeitner, Rachel C.; Goldstone, Jared V.; McArthur, Andrew G.; Hahn, Mark E.; Woods Hole Oceanographic Institution; University of Alabama TuscaloosaNF-E2-related factor 2 (NRF2; also called NFE2L2) and related NRF family members regulate antioxidant defenses by activating gene expression via antioxidant response elements (AREs), but their roles in embryonic development are not well understood. We report here that zebrafish (Danio rerio), an important developmental model species, possesses six nrf genes, including duplicated nrf1 and nrf2 genes. We cloned a novel zebrafish nrf2 paralog, nrf2b. The predicted Nrf2b protein sequence shares several domains with the original Nrf2 (now Nrf2a) but lacks the Neh4 transactivation domain. Zebrafish-human comparisons demonstrate conserved synteny involving nrf2 and hox genes, indicating that nrf2a and nrf2b are co-orthologs of human NRF2. nrf2a and nrf2b displayed distinct patterns of expression during embryonic development; nrf2b was more highly expressed at all stages. Embryos in which Nrf2a expression had been knocked down with morpholino oligo-nucleotides were more sensitive to tert-butylhydroperoxide but not tert-butylhydroquinone, whereas knockdown of Nrf2b did not affect sensitivity of embryos to either chemical. Gene expression profiling by microarray identified a specific role for Nrf2b as a negative regulator of several genes, including p53, cyclin G1, and heme oxygenase 1, in embryos. Nrf2a and Nrf2b exhibited different mechanisms of cross-talk with the Ahr2 signaling pathway. Together, these results demonstrate distinct roles for nrf2a and nrf2b, consistent with sub-function partitioning, and identify a novel negative regulatory role for Nrf2b during development. The identification of zebrafish nrf2 co-orthologs will facilitate new understanding of the multiple roles of NRF2 in protecting vertebrate embryos from oxidative damage.Item Purification of full-length VP22 from cells infected with HSV-1: A two-pronged approach for the solubilization and purification of viral proteins for use in biochemical studies(Elsevier, 2012) Dewberry, Ebony J.; Dunkerley, Eric; Duffy, Carol; University of Alabama TuscaloosaVP22, encoded by the U(L)49 gene, is one of the most abundant proteins of the herpes simplex virus type 1 (HSV-1) tegument and has been shown to be important for virus replication and spread. However, the exact role(s) played by VP22 in the HSV-1 replication cycle have yet to be delineated. The lack of a procedure to purify full-length VP22 has limited molecular studies on VP22 function. A procedure was developed for the purification of soluble, full-length VP22 from cells infected with HSV-1. A recombinant virus encoding His-tagged VP22 was generated and found to express VP22 at levels comparable to the wild type virus upon infection of Vero cells. By experimenting with a wide variety of cell lysis buffer conditions, several buffers that promote the solubility of full-length VP22 were identified. Buffers that gave the highest levels of solubility were then used in immobilized metal ion affinity chromatography experiments to identify conditions that provided the greatest level of VP22 binding and recovery from cobalt and nickel affinity resins. Using this strategy soluble, full-length VP22 was purified from cells infected with HSV-1. (C) 2012 Elsevier B.V. All rights reserved.Item A new species of leopard frog (Anura: Ranidae) from the urban northeastern US(Elsevier, 2012) Newman, Catherine E.; Feinberg, Jeremy A.; Rissler, Leslie J.; Burger, Joanna; Shaffer, H. Bradley; University of California Davis; Rutgers State University New Brunswick; University of Alabama TuscaloosaPast confusion about leopard frog (genus Rana) species composition in the Tri-State area of the US that includes New York (NY), New Jersey (NJ), and Connecticut (CT) has hindered conservation and management efforts, especially where populations are declining or imperiled. We use nuclear and mitochondrial genetic data to clarify the identification and distribution of leopard frog species in this region. We focus on four problematic frog populations of uncertain species affiliation in northern NJ, southeastern mainland NY, and Staten Island to test the following hypotheses: (1) they are conspecific with Rana sphenocephala or R. pipiens, (2) they are hybrids between R. sphenocephala and R. pipiens, or (3) they represent one or more previously undescribed cryptic taxa. Bayesian phylogenetic and cluster analyses revealed that the four unknown populations collectively form a novel genetic lineage, which represents a previously undescribed cryptic leopard frog species, Rana sp. nov. Statistical support for R. sp. nov. was strong in both the Bayesian (pp = 1.0) and maximum-likelihood (bootstrap = 99) phylogenetic analyses as well as the Structure cluster analyses. While our data support recognition of R. sp. nov. as a novel species, we recommend further study including fine-scaled sampling and ecological, behavioral, call, and morphological analyses before it is formally described. (C) 2012 Elsevier Inc. All rights reserved.Item Myristoylation Exerts Direct and Allosteric Effects on G alpha Conformation and Dynamics in Solution(American Chemical Society, 2012) Preininger, Anita M.; Kaya, Ali I.; Gilbert, James A., III; Busenlehner, Laura S.; Armstrong, Richard N.; Hamm, Heidi E.; Vanderbilt University; University of Alabama TuscaloosaCoupling of heterotrimeric G proteins to activated G protein-coupled receptors results in nucleotide exchange on the G alpha subunit, which in turn decreases its affinity for both G beta gamma and activated receptors. N-Terminal myristoylation of G alpha subunits aids in membrane localization of inactive G proteins. Despite the presence of the covalently attached myristoyl group, G alpha proteins are highly soluble after GTP binding. This study investigated factors facilitating the solubility of the activated, myristoylated protein. In doing so, we also identified myristoylation-dependent differences in regions of G alpha known to play important roles in interactions with receptors, effectors, and nucleotide binding. Amide hydrogen deuterium exchange and site-directed fluorescence of activated proteins revealed a solvent-protected amino terminus that was enhanced by myristoylation. Furthermore, fluorescence quenching confirmed that the myristoylated amino terminus is in proximity to the Switch H region in the activated protein. Myristoylation also stabilized the interaction between the guanine ring and the base of the alpha 5 helix that contacts the bound nucleotide. The allosteric effects of myristoylation on protein structure, function, and localization indicate that the myristoylated terminus of G alpha(i) functions as a myristoyl switch, with implications for myristoylation in the stabilization of nucleotide binding and in the spatial regulation of G protein signaling.Item Biodegradable Orthopedic Magnesium-Calcium (MgCa) Alloys, Processing, and Corrosion Performance(MDPI, 2012) Salahshoor, Meisam; Guo, Yuebin; University of Alabama TuscaloosaMagnesium-Calcium (Mg-Ca) alloy has received considerable attention as an emerging biodegradable implant material in orthopedic fixation applications. The biodegradable Mg-Ca alloys avoid stress shielding and secondary surgery inherent with permanent metallic implant materials. They also provide sufficient mechanical strength in load carrying applications as opposed to biopolymers. However, the key issue facing a biodegradable Mg-Ca implant is the fast corrosion in the human body environment. The ability to adjust degradation rate of Mg-Ca alloys is critical for the successful development of biodegradable orthopedic implants. This paper focuses on the functions and requirements of bone implants and critical issues of current implant biomaterials. Microstructures and mechanical properties of Mg-Ca alloys, and the unique properties of novel magnesium-calcium implant materials have been reviewed. Various manufacturing techniques to process Mg-Ca based alloys have been analyzed regarding their impacts on implant performance. Corrosion performance of Mg-Ca alloys processed by different manufacturing techniques was compared. In addition, the societal and economical impacts of developing biodegradable orthopedic implants have been emphasized.