Biochemistry, Biophysics, and Structural Biology Commons^™

Evidence For Direct Roles Of Two Additional Factors, Secp43 And Soluble Liver Antigen, In The Selenoprotein Synthesis Machinery, Xue-Ming Xu, Heiko Mix, Bradley A. Carlson, Paula J. Grabowski, Vadim N. Gladyshev, Marla J. Berry, Dolph L. Hatfield

Vadim Gladyshev Publications

Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA ^[Ser]Sec, but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2’-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA ^[Ser]Sec, and consequently reduced glutathione peroxidase …

Computer-Assisted Docking Of Flavodoxin With The Atp:Co(I)Rrinoid Adenosyltransferase (Coba) Enzyme Reveals Residues Critical For Protein-Protein Interactions But Not For Catalysis*, Nicole R. Buan, Jorge C. Escalante-Semerena

Department of Biochemistry: Faculty Publications

The activity of the housekeeping ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica sv. Typhimurium is required to adenosylate de novo biosynthetic intermediates of adenosylcobalamin and to salvage incomplete and complete corrinoids from the environment of this bacterium. In vitro, reduced flavodoxin (FldA) provides an electron to generate the co(I)rrinoid substrate in the CobA active site. To understand how CobAand FldA interact, a computer model of aCobA∙FldA complex was generated. This model was used to guide the introduction of mutations into CobA using site-directed mutagenesis and the synthesis of a peptide mimic of FldA. Residues Arg-9 and Arg-165 of CobA …

[Accepted Article Manuscript Version (Postprint)] Identification And Functional Characterization Of Arabidopsis Peroxin4 And The Interacting Protein Peroxin22, Bethany Zolman, Melanie Monroe-Augustus, Illeana Silva, Bonnie Bartel

Biology Department Faculty Works

Peroxins are genetically defined as proteins necessary for peroxisome biogenesis. By screening for reduced response to indole-3-butyric acid, which is metabolized to active auxin in peroxisomes, we isolated an Arabidopsis thaliana peroxin4 (pex4) mutant. This mutant displays sucrose-dependent seedling development and reduced lateral root production, characteristics of plant peroxisome malfunction. We used yeast two-hybrid analysis to determine that PEX4, an apparent ubiquitinconjugating enzyme, interacts with a previously unidentified Arabidopsis protein, PEX22. A pex4 pex22 double mutant enhanced pex4 defects, confirming that PEX22 is a peroxin. Expression of both Arabidopsis genes together complemented yeast pex4 or pex22 mutant defects, whereas expression …

Mechanisms Of Hbo-Induced Neuroprotection In A Neonatal Hypoxia-Ischemia Model, John Winter Calvert

Loma Linda University Electronic Theses, Dissertations & Projects

With an incidence approaching 1/4000 live births and as high as 60% in low birth weight infants, cerebral hypoxia-ischemia during the perinatal period is the single most important cause of acute mortality and chronic disability in newborns. Herein, we tested the hypothesis that following a hypoxic-ischemic insult hyperbaric oxygen (HBO) treatment can restore high energy metabolite levels in the affected regions of the brain and through this increase in energy levels ameliorate the spread of cell death following the insult. We also investigated if an additional mechanism by which HBO affords its neuroprotection is by altering the expression of the …

Different Catalytic Mechanisms In Mammalian Selenocysteine- And Cysteine-Containing Methionine-R-Sulfoxide Reductases, Hwa-Young Kim, Vadim Gladyshev

Vadim Gladyshev Publications

Selenocysteine (Sec) is found in active sites of several oxidoreductases in which this residue is essential for catalytic activity. However, many selenoproteins have fully functional orthologs, wherein cysteine (Cys) occupies the position of Sec. The reason why some enzymes evolve into selenoproteins if the Cys versions may be sufficient is not understood. Among three mammalian methionine-R-sulfoxide reductases (MsrBs), MsrB1 is a Sec-containing protein, whereas MsrB2 and MsrB3 contain Cys in the active site, making these enzymes an excellent system for addressing the question of why Sec is used in biological systems. In this study, we found that residues, which are …

Process For The Preparation Of Citalopram Intermediate, Aslam A. Malik, Hasan Palandoken, Joy A. Stringer, Dershing Huang, Antonio Romero, Olivier Dapremont

Chemistry and Biochemistry

The present invention provides, inter alia, a novel process for the preparation of Citalopram, a known antidepressant.

Loss Of Editing Activity During The Evolution Of Mitochondrial Phenylalanyl-Trna Synthetase, Hervé Roy, Jiqiang Ling, Juan D. Alfonzo, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNA^Phe. Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNA^Phe, an activity enhanced in …

The Role Of Phosducin-Like Protein And The Cytosolic Chaperonin Cct In G Beta Gamma Dimer Assembly, Ting Hu

Theses and Dissertations

Phosducin-like protein (PhLP), a G protein beta gamma subunit dimer binder and G protein signaling regulator, was suggested to regulate the activity of cytosolic chaperonin CCT by their high affinity interaction. In the present study, the three-dimensional structure of PhLP:CCT complex has been solved by cryoelectron microscopy. PhLP was found to bind only one of the chaperonin rings with both N- and C-terminal domains. It spans the central folding cavity of CCT and interacts with two opposite sides of the top apical region, inducing the constraining of the entry of the folding cavity. These findings support a putative role of …

Palladium-Imidazolium Carbene Catalyzed Heck Coupling Reactions And Synthesis Of A Novel Class Of Fluoroanthracenylmethyl Ptc Catalysts, Jiuqing Zhang

Theses and Dissertations

Palladium catalyzed Heck coupling with aryl and alkenyl halides has become a powerful means of carbon-carbon bond formation. This standard synthetic method has been developed to a high level of utility using various catalysts, conditions and substrates. Yet significant drawbacks remain, including poor reactivity, the need for high temperatures and base, limited substrate generality, and selectivity. Mixed products often suffer from olefin migration following insertion. N-Heterocyclic carbenes (NHC) have proven to be electron-rich donors which provide higher stability and reactivity than phosphines. In a previous paper reported by our research group the imidazolium-palladium carbene has proven to be highly efficient …

A Novel Cysteine-Rich Domain Of Sep15 Mediates The Interaction With Udp-Glucose:Glycoprotein Glucosyltransferase, Vyacheslav M. Labunskyy, Andrew D. Ferguson, Dmitri E. Fomenko, Yogarany Chelliah, Dolph L. Hatfield, Vadim N. Gladyshev

Vadim Gladyshev Publications

Selenium is an essential trace element with potent cancer prevention activity in mammals. The 15-kDa selenoprotein (Sep15) has been implicated in the chemopreventive effect of dietary selenium. Although the precise function of Sep15 remains elusive, Sep15 copurifies with UDP-glucose:glycoprotein glucosyltransferase (GT), an essential regulator of quality control mechanisms within the endoplasmic reticulum. Recent studies identified two GT and two Sep15 homologues in mammals. We characterize interactions between these protein families in this report. Sep15 and GT form a tight 1:1 complex, and these interactions are conserved between mammals and fruit flies. In mammalian cells, Sep15 co-immunoprecipitates with both GT isozymes. …

Performance Of Binary-Encounter-Bethe (Beb) Theory For Electron-Impact Ionization Cross Sections Of Molecules Containing Heavy Elements (Z > 10), Gregory E. Scott, Karl K. Irikura

Chemistry and Biochemistry

The binary-encounter-Bethe (BEB) theory developed by Kim and coworkers has been successful for computing electron-impact ionization cross sections of many molecules. However, some recent publications have stated that BEB theory performs poorly for molecules that contain heavier elements such as chlorine and sulfur. We have found that the BEB calculations in those publications were performed incorrectly. When performed correctly, BEB predictions are as good for heavy-element molecules as for light-element molecules. We recommended recently that an alternative, less-confusing procedure be used for molecules that contain heavier elements. The alternative procedure, based upon effective core potentials (ECPs), does not require explicit …

Total Syntheses Of (+)-Geldanamycin, (-)-Ragaglitazar, And (+)-Kurasoin A And Phase-Transfer-Catalyzed Asymmetric Alkylation, Erik J. Hicken

Theses and Dissertations

Geldanamycin possesses various biological activities as seen in the NCI 60 cell line panel (13 nM avg., 70 nM SKBr-3 cells). The predominant mode of action providing these unique results arises from the ability of geldanamycin (GA) to bind to the chaperone heat shock protein 90 (Hsp90). Despite its complicated functionality, the first total synthesis of GA was accomplished, which included two new reactions developed specifically to address the stereochemical features. The final step in the synthesis of GA was a demethylation-oxidation sequence to generate the desired para-quinone. This step could only be accomplished with HNO3/AcOH, producing GA in …

Crystal Structures Of Oxidized And Reduced Mitochondrial Thioredoxin Reductase Provide Molecular Details Of The Reaction Mechanism, Ekaterina I. Biterova, Anton A. Turanov, Vadim N. Gladyshev, Joseph J. Barycki

Vadim Gladyshev Publications

Thioredoxin reductase (TrxR) is an essential enzyme required for the efficient maintenance of the cellular redox homeostasis, particularly in cancer cells that are sensitive to reactive oxygen species. In mammals, distinct isozymes function in the cytosol and mitochondria. Through an intricate mechanism, these enzymes transfer reducing equivalents from NADPH to bound FAD and subsequently to an active-site disulfide. In mammalian TrxRs, the dithiol then reduces a mobile C-terminal selenocysteine- containing tetrapeptide of the opposing subunit of the dimer. Once activated, the C-terminal redox center reduces a disulfide bond within thioredoxin. In this report, we present the structural data on a …

Specific Heat And Thermodynamic Properties Of Metallic Systems: Instrumentation And Analysis, Brian E. Lang

Theses and Dissertations

A small-scale adiabatic calorimeter has been constructed as part of a larger project to study nano-particles and to facilitate specific heat measurements on samples where it is difficult to obtain enough material to run on the current large-scale adiabatic apparatus. This calorimeter is designed to measure sample sizes of less than 0.8 cc over a temperature range from 13 K to 350 K. Specific heat results on copper, sapphire, and benzoic acid show the accuracy of the measurements to be better than ±0.4% for temperatures higher than 50 K. The reproducibility of these measurements is generally better than ±0.25%. Experimental …

Microcapsule Biosensors Based On Competitive Binding And Fluorescence Resonance Energy Transfer Assays, Swetha Chinnayelka

Doctoral Dissertations

Fluorescent sensing systems offer the potential for minimally invasive monitoring with implantable devices, but they require carrier technologies that provide suitable immobilization, accessibility, and biocompatibility while maintaining adequate response characteristics. Towards the development of this goal, a general design of a biosensor with the capability of detecting different metabolites was investigated. The approach is based on the encapsulation of a competitive binding assay in microcapsules and monitoring the changes in fluorescence resonance energy transfer (FRET) in the presence of analyte. To experimentally demonstrate this type of sensing system, glucose was chosen as the model target analyte. The design, fabrication, and …

Chromosomal Localization Of The Islet Neogenesis Associated Protein (Ingap) Gene In Syrian Hamster By Tyramide Signal Amplification-Fluorescence In Situ Hybridization (Tsa-Fish), Sallie A. Smith

Biological Sciences Theses & Dissertations

Diabetes mellitus is a group of conditions characterized by hyperglycemia due to an inability to produce or properly utilize insulin. The majority of cases fall into two categories, Type I and Type 2. Type I results from the autoimmune destruction of pancreatic β-cells of the islets. The beta cells are the exclusive source of insulin and the patient becomes entirely dependent on exogenous insulin to survive. Patients with Type 2 are distinguished by insulin resistance, a condition that develops due to the inability of the body to effectively use the insulin being produced. The β-cells gradually lose their ability to …

A Facile Synthesis Of (Tert-Alkoxy)Amines, Hasan Palandoken, Chris M. Bocian, Michelle R. Mccombs, Michael H. Nantz

Chemistry and Biochemistry

Tertiary alcohols react with stoichiometric BF₃·Et₂O and N-hydroxyphthalimide to yield N-alkoxyphthalimides. Subsequent hydrazinolyses afford the title compounds.

Polymer Microchips For Capillary Electrophoresis And Electric Field Gradient Focusing Of Biomolecules, Ryan Thomas Kelly

Theses and Dissertations

Polymeric materials have seen increasing use as microfluidic device substrates due to their low cost and the simplicity of templated fabrication procedures. I showed that poly(methyl methacrylate) (PMMA) microdevices could be enclosed in a boiling water bath, which allowed the seal to form more quickly than in conventional approaches, and enabled microchannels to remain hydrated throughout the bonding process. Microchip capillary electrophoresis (µ-CE) devices were fabricated using water-based enclosure, and a mixture of fluorescently labeled amino acids was separated in 30 s in these microchips. To create more robust capillary electrophoresis (CE) microdevices with improved separation performance, phase-changing sacrificial materials …

Fractals Analysis Of Cardiac Arrhythmias, Mohammed Saeed

Department of Biological & Biomedical Sciences

Heart rhythms are generated by complex self-regulating systems governed by the laws of chaos. Consequently, heart rhythms have fractal organization, characterized by self-similar dynamics with long-range order operating over multiple time scales. This allows for the self-organization and adaptability of heart rhythms under stress. Breakdown of this fractal organization into excessive order or uncorrelated randomness leads to a less-adaptable system, characteristic of aging and disease. With the tools of nonlinear dynamics, this fractal breakdown can be quantified with potential applications to diagnostic and prognostic clinical assessment. In this paper, I review the methodologies for fractal analysis of cardiac rhythms and …

Structure Elucidation Of Bioactive Compounds Isolated From Endophytes Of Alstonia Scholaris And Acmena Graveolens, Nicholas James Hundley

Theses and Dissertations

Alstonia scholaris is an evergreen tree native to Southeast Asia and Australia. It is commonly used as a medicinal plant throughout these regions. In the present study, an endophyte of the genus Xylaria was isolated from a stem of Alstonia scholaris, its mycelia and exudate extracted, and the extract assayed for growth inhibition of HeLa cancer cells in vitro. Several known compounds were isolated and identified based on NMR, infrared, and mass spectral data. The compounds identified are 19,20-epoxycytochalasin C; 19,20epoxycytochalasin D; and xylobovide. Two other compounds, fusaric acid and dehydrofusaric acid, were discovered in an endophyte of the Hypocreales …

An Acyl Radical Cascade Model For The Total Synthesis Of Lyconadin A, Seth W. Grant

Theses and Dissertations

Lyconadin A (1) is a structurally unique Lycopodium alkaloid with antitumor properties, isolated from the club moss Lycopodium complanatum. We are developing a synthetic route to 1 based on a novel 7-exo-trig/6-exo-trig acyl radical cascade cyclization. The synthesis of model acyl radical cascade precursor 23 will be presented. Key features of this synthesis include the suppression of competing elimination during the alkylation of a hindered phenethyl bromide and the use of a lactone as a precursor to a compound bearing two differentially protected primary alcohols. An account of our studies on the model acyl radical cascade cyclization (23 to 24 …

Effect Of Melatonin And Dopamine In Site Specific Phosphorylation Of Phosducin In Intact Retina, Arinzechukwu Okere Nkemdirim

Theses and Dissertations

Phosducin (Pdc) is a 28 kDa binding partner for the G protein beta gamma subunit dimer (G-beta-gamma) found abundantly in the photoreceptor cells of the retina and pineal gland. In the retina, light-dependent changes in cAMP and Ca2+ control the phosphorylation of Pdc at serine 73 and 54, respectively, which in turn controls the binding of Pdc to G protein beta gamma subunit dimer . G protein beta gamma subunit dimer binding has been proposed to facilitate light-driven transport of G protein beta gamma subunit dimer from the site of phototransduction in the outer segment of the photoreceptor cell to …

Charge-Switch Nucleotides, John G. K. Williams, Gregory R. Bashford, Jiyan Chen, Dan Draney, Nara Narayanan, Bambi L. Reynolds, Pamela Sheaff

Biomedical Imaging and Biosignal Analysis Laboratory

The present invention provides compounds, methods and systems for sequencing nucleic acid using single molecule detection. Using labeled NPs that exhibit charge-switching behavior, single-molecule DNA sequencing in a microchannel sorting system is realized. In operation, sequencing products are detected enabling real-time sequencing as successive detectable moieties flow through a detection channel. By electrically sorting charged molecules, the cleaved product molecules are detected in isolation Without interference from unincorporated NPs and Without illuminating the polymerase-DNA complex.

Post-Translational Regulation Of Adipose Differentiation-Related Protein By The Ubiquitin/Proteasome Pathway, John Tansey

Chemistry Faculty Scholarship

Adipose differentiation-related protein (ADRP) is localized to lipid droplets in most mammalian cells. ADRP, proposed to regulate
fatty acid mobilization and lipid droplet formation, is linked to lipid accumulation in foam cells of human atherosclerotic
lesions. In this report, we show that ADRP protein accumulates in Chinese hamster ovary fibroblastic cells cultured in the
presence of oleic acid but is destabilized when fatty acid sources are removed from culture serum. The latter effect was blocked
by the proteasome inhibitor MG132, whereas inhibitors of other proteolytic processes were ineffective. Pulse-chase experiments
confirmed that ADRP degradation is inhibited by MG132. Conditions that …

Isolation, Purification, And Chemical Characterization Of The Dihydroxamate-Type Siderophore, "Schizokinen," Produced By Rhizobium Leguminosarum Iari 917., Erin P. Storey

Electronic Theses and Dissertations

Iron is essential to the majority of microorganisms; it is an important cofactor in many cellular processes and enzymes. However in an aerobic environment and at biological pH, iron is primarily found as insoluble oxyhydroxides and is unavailable to microorganisms. Many bacteria have the ability to produce siderophores, low molecular weight compounds that have a high affinity for Fe³⁺. Siderophores are part of a multi-component system that actively transports the iron-siderophore complex into the cytoplasm. Rhizobia are characterized by their ability to form symbiotic relationships with leguminous plants, where they can fix nitrogen for the host plant and …

The Clearance Of Protein-Bound Solutes By Hemofiltration And Hemodiafiltration, Timothy W. Meyer, Jason L. Walther, Maria Enrica Pagtalunan, Andres W. Martinez, Ali Torkamani, Patrick D. Fong, Natalie S. Recht, Channing R. Robertson, Thomas H. Hostetter

Chemistry and Biochemistry

Background. Hemofiltration in the form of continuous venovenous hemofiltration (CVVH) is increasingly used to treat acute renal failure. Compared to hemodialysis, hemofiltration provides high clearances for large solutes but its effect on protein-bound solutes has been largely ignored.

Methods. Standard clinical systems were used to remove test solutes from a reservoir containing artificial plasma. Clearances of the protein-bound solutes phenol red (C_PR) and indican (C_IN) were compared to clearances of urea (C_UREA) during hemofiltration and hemodiafiltration. A mathematical model was developed to predict clearances from values for plasma flow Q_p, …

Fluorescent Photoinduced Electron Transfer (Pet) Cation Sensors Derived From Natural Products, Kenny Loo

Theses, Dissertations and Culminating Projects

Two new second generation fluorescent switches that are based on the chromophore-spacer-receptor architecture are reported in this thesis. The fluorescence modulation of these switches is a result of quenching or initiating photoinduced electron transfer (PET) between the chromophore and the receptors due to cation binding. These two molecules utilize an alkaloid, anabasine, as the proton receptor, and function as fluorescent off-on-off switches with decreasing pH. One of these molecules also utilizes a coumarin as the chromophore, making it the first rationally designed fluorescent PET system based on natural products. Further studies of two well-known first generation fluorescent switches to achieve …

Regulatable Gene Expression Systems For Gene Therapy Applications: Progress And Future Challenges, Shyam Goverdhana, Mariana Puntel, Weidong Xiong, Jeffrey Zirger, Carlos Barcia, James Curtin, Eric Soffer, Sonali Mondkar, Gwendalyn King, Jinwei Hu, Marianela Candolfi, Diane Greengold, Pedro Lowenstein, Maria Castro

Articles

Gene therapy aims to revert diseased phenotypes by the use of both viral and nonviral gene delivery systems. Substantial progress has been made in making gene transfer vehicles more efficient, less toxic, and nonimmunogenic and in allowing long-term transgene expression. One of the key issues in successfully implementing gene therapies in the clinical setting is to be able to regulate gene expression very tightly and consistently as and when it is needed. The regulation ought to be achievable using a compound that should be nontoxic, be able to penetrate into the desired target tissue or organ, and have a half-life …

A Role For Mitogen-Activated Protein Kinase (Erk1/2) Activation And Non-Selective Pore Formation In P2x7 Receptor-Mediated Thymocyte Death, Rodolphe Auger, Iris Motta, Karim Benihoud, David M. Ojcius, Jean M. Kanellopoulos

All Dugoni School of Dentistry Faculty Articles

Extracellular ATP (ATPe) binds to P2X7 receptors (P2X7R) expressed on the surface of cells of hematopoietic lineage, including murine thymocytes. Activation of P2X7R by ATPe results in the opening of cation-specific channels, and prolonged ATPe exposure leads to the formation of non-selective pores enabling transmembrane passage of solutes up to 900 Da. In the presence of ATPe, P2X7R-mediated thymocyte death is due primarily to necrosis/lysis and not apoptosis, as measured by the release of lactate dehydrogenase indicative of a loss of plasma membrane integrity. The present study is focused on the identification of P2X7R signaling mediators in ATP-induced thymocyte necrosis/lysis. …

Lack Of Association Of Methylenetetrahydrofolate Reductase 677c>T Mutation With Coronary Artery Disease In A Pakistani Population, M Perwaiz Iqbal, Tasneem Fatima, Siddiqa Parveen, Farzana A. Yousuf, Majid Shafiq, Naseema Mehboobali, Abrar H. Khan, Iqbal Azam, Philippe M. Frossard

Department of Biological & Biomedical Sciences

Pakistanis belong to the South Asian population which has the highest known rate of coronary artery disease. Folic acid deficiency also appears to be highly prevalent in this population. Methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism decreases the activity of this enzyme and can be associated with mild to moderate hyperhomocysteinemia in homozygotes, particularly when there is folic acid deficiency, as well as with coronary artery disease. To assess the value of genotyping the MTHFR 677C>T dimorphism, we carried out a case-control study of dimorphism 677C>T for putative association with myocardial infarction (MI) among Pakistani nationals. We investigated a …