Digital Commons Network^™

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 …

Evolution Of Selenium Utilization Traits, Hector Romero, Yan Zhang, Vadim Gladyshev, Gustavo Salinas

Vadim Gladyshev Publications

Background: The essential trace element selenium is used in a wide variety of biological processes. Selenocysteine (Sec), the 21st amino acid, is co-translationally incorporated into a restricted set of proteins. It is encoded by an UGA codon with the help of tRNA^Sec (SelC), Sec-specific elongation factor (SelB) and a cis-acting mRNA structure (SECIS element). In addition, Sec synthase (SelA) and selenophosphate synthetase (SelD) are involved in the biosynthesis of Sec on the tRNA^Sec. Selenium is also found in the form of 2-selenouridine, a modified base present in the wobble position of certain tRNAs, whose synthesis is …

A Modified Poisson–Boltzmann Analysis Of The Capacitance Behavior Of The Electric Double Layer At Low Temperatures, Douglas Henderson, L. B. Bhuiyan, C. W. Outhwaite

Faculty Publications

The modified Poisson–Boltzmann theory is used to analyze the anomalous behavior of the electric double layer capacitance for small surface charge at low temperatures and densities. Good agreement is found with simulation and recent density-functional theory results. Negative adsorption is also found in line with theory and simulation. An unsatisfactory feature is the relatively poor structure in this region due to the inherent approximations in the theory. This feature is unimportant in relation to the capacitance results but has implications when calculating adsorption properties.

Hard-Sphere Radial Distribution Function Again, Andrij Trokhymchuk, Douglas Henderson, Ivo Nezbeda, Jan Jirsak

Faculty Publications

A theoretically based closed-form analytical equation for the radial distribution function, g(r), of a fluid of hard spheres is presented and used to obtain an accurate analytic representation. The method makes use of an analytic expression for the short- and long-range behaviors of g(r), both obtained from the Percus-Yevick equation, in combination with the thermodynamic consistency constraint. Physical arguments then leave only three parameters in the equation of g(r) that are to be solved numerically, whereas all remaining ones are taken from the analytical solution of the Percus-Yevick equation.

Mammalian Selenoprotein Thioredoxin-Glutathione Reductase, Dan Su, Sergey V. Novoselov, Qi-An Sun, Mohamed E. Moustafa, You Zhou, Richard Oko, Dolph L. Hatfield, Vadim N. Gladyshev

Vadim Gladyshev Publications

Thioredoxin reductases (TRs) are important redox regulatory enzymes, which control the redox state of thioredoxins. Mammals have cytosolic and mitochondrial TRs, which contain an essential selenocysteine residue and reduce cytosolic and mitochondrial thioredoxins. In addition, thioredoxin/glutathione reductase (TGR) was identified, which is a fusion of an N-terminal glutaredoxin domain and the TR module. Here we show that TGR is expressed at low levels in various tissues but accumulates in testes after puberty. The protein is particularly abundant in elongating spermatids at the site of mitochondrial sheath formation but is absent in mature sperm. We found that TGR can catalyze isomerization …

Angiotensin Ii-Mediated Regulation Of The Human Angiotensin Ii Type 1 Receptor Gene, Xylophone Vijai Aasee Victor

Theses and Dissertations

The physiological responses of angiotensin II (Ang II) are mediated across the cell membrane through the angiotensin II type 1 receptor (AT1R), a heptahelical membrane protein coupled to trimeric G-proteins on the cytosolic side. AT1R on binding its ligand, Ang II, leads to downregulation of cell-surface receptor and also its mRNA. We have investigated whether the 3'- and 5'-untranslated regions of the human AT1R mRNA mediate the degradation of hAT1R mRNA by post-transcriptional mechanisms in human adrenocortical carcinoma cell line (H295R cells). Protein kinase C (PKC) activator, phorbol-12-myristate-13-acetate (PMA), showed that the downregulation of hAT1R mRNA is mediated by the …

Development Of A Model For The Kinetics And Mechanism Of Nitrogenase, Phillip E. Wilson

Theses and Dissertations

Nitrogenase has a central role in the global nitrogen cycle as the enzyme that catalyzes the reduction of atmospheric N2 to NH3. Fixed nitrogen is generally limiting in the environment and in agriculture, so nitrogenase has received much attention as an alternative to nitrogen fertilizers. Characterizing the mechanism of nitrogenase is the goal of this work. The molybdenum nitrogenase enzyme system is comprised of the MoFe protein and the Fe protein. Interactions between these proteins and nucleotides are crucial to catalysis. An important approach to characterize these interactions is to correlate the kinetics of nitrogenase catalysis to a mechanism based …

The Cell Cycle–Regulated Genes Of Schizosaccharomyces Pombe, Anna Oliva, Adan Rosebrock, Francisco Ferrezuelo, Haiying Chen, Saumyadipta Pyne, Steve Skiena, Bruce Futcher, Janet Leatherwood

Department of Molecular Genetics and Microbiology Faculty Publications

Many genes are regulated as an innate part of the eukaryotic cell cycle, and a complex transcriptional network helps enable the cyclic behavior of dividing cells. This transcriptional network has been studied in Saccharomyces cerevisiae (budding yeast) and elsewhere. To provide more perspective on these regulatory mechanisms, we have used microarrays to measure gene expression through the cell cycle of Schizosaccharomyces pombe (fission yeast). The 750 genes with the most significant oscillations were identified and analyzed. There were two broad waves of cell cycle transcription, one in early/mid G2 phase, and the other near the G2/M transition. The early/mid G2 …

An Analysis Of The Chemical Processes In The Smoke Plume From A Savanna Fire, Jorg Trentmann, Robert J. Yokelson, Peter V. Hobbs, Tanja Winterrath, Ted J. Christian, Meinrat O. Andreae, Sherri A. Mason

Chemistry and Biochemistry Faculty Publications

[1] Photochemistry in young plumes from vegetation fires significantly transforms the initial fire emissions within the first hour after the emissions are injected into the atmosphere. Here we present an investigation of field measurements obtained in a smoke plume from a prescribed savanna fire during the SAFARI 2000 field experiment using a detailed photochemical box-dilution model. The dilution used in the model simulations was constrained by measurements of chemically passive tracers (e.g., CO) near and downwind of the fire. The emissions of the dominant carbonaceous compounds, including oxygenated ones, were taken into account. The field measurements revealed significant production of …

Carbon Cycling In Lake Superior, N. R. Urban, M. T. Auer, Sarah A. Green, X. Lu, D. S. Apul, K. D. Powell, L. Bub

Department of Chemistry Publications

Carbon (C) cycling in Lake Superior was studied within the Keweenaw Interdisciplinary Transport Experiment in Superior (KITES) project to assess (1) whether the lake is net heterotrophic, (2) sources, sinks and residence time for dissolved organic carbon (DOC), (3) importance of terrigenous organic C subsidies, and (4) factors limiting C flow through bacteria. During 3 years of fieldwork, measurements were made of spatial and temporal distributions of C pools and rates of photosynthesis, community respiration, and bacterial production. Measurements were made of the composition of dissolved organic matter (DOM), rates of DOM photolysis, lability of DOM toward microbial consumption, and …

A Critical Role For Kalirin In Ngf Signaling Through Trka, Kausik Chakrabarti, Rong Lin, Noraisha I. Schiller, Yanping Wang, David Koubi, Ying-Xin Fan, Brian B. Rudkin, Gibbes R. Johnson, Martin R. Schiller

Life Sciences Faculty Research

Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low …

A Revised Nomenclature For Mammalian Acyl-Coa Thioesterases/Hydrolases, Mary Hunt, Junji Yamada, Lois Maltais, Mathew Wright, Ernesto Podesta, Stefan Alexson

Articles

Acyl-CoA thioesterases, also known as acyl-CoA hydrolases, are a group of enzymes that hydrolyze CoA esters such as acyl-CoAs (saturated, unsaturated, branched chain), bile acid-CoAs, CoA esters of prostaglandins etc, to the corresponding free acid and coenzyme A. There is however significant confusion regarding the nomenclature of these genes. In agreement with the HUGO Gene Nomenclature Committee (HGNC) and the Mouse Genomic Nomenclature Committee (MGNC), a revised nomenclature for mammalian acyl-CoA thioesterases/hydrolases has been suggested for the 12 member family. The family root symbol is ACOT, with human genes named ACOT1-12, and rat and mouse named Acot1-12. Several of the …

Glycoprotein Gp130 Of Dictyostelium Discoideum Influences Macropinocytosis And Adhesion, Catherine P. Chia, Sujatha Gomathinayagam, Robert J. Schmaltz, Laura K. Smoyer

Department of Biochemistry: Faculty Publications

Glycoprotein gp130, found on the plasma membrane of Dictyostelium discoideum amoebae, was postulated previously to play a role in phagocytosis. The gene for gp130 was cloned and when translated, yielded a 768 amino acid preproprotein of 85.3 kDa. It had nearly 40% similarity to the 138 kDa family of glycoproteins implicated in sexual cell fusion during macrocyst formation in D. discoideum. The difference between the calculated size and observed Mr of 130 kDa on protein gels likely was due to N-glycosylation that was confirmed by lectin blots. Consistent with its surface-exposure, an antibody raised against recombinant protein stained …