Life Sciences Commons^™

Molecular Modeling Of Proteins And Peptides Related To Cell Attachment In Vivo And In Vitro, Wanhua Zhao

Doctoral Dissertations

Polypeptides constitute half of the dry mass of the cell, they form the bulk of the extracellular matrix (ECM), and they are a common element of extra- and intracellular signaling pathways. There is increasing interest in the development of computational methods in polypeptide and protein engineering on all length scales. This research concerns the development of computational methods for study of polypeptide interactions related to cell attachment in vivo and in vitro.

Polypeptides are inherently biocompatible, and an astronomical range of unique sequences can be designed and realized in massive quantities by modern methods of synthesis and purification. These …

Extracellular Superoxide Dismutase Protects Cardiomyocytes During Hyperoxia, Mervat Elsayed Ali

Theses and Dissertations in Biomedical Sciences

Reactive oxygen species are implicated in tissue damage in many cardiovascular diseases. The current study was designed to test the hypothesis that exposure to high inspired oxygen concentrations (100%) damages cardiac mitochondria and that a biogenic response is needed for cell survival. The study was done in mice with a transgene (TG) for extracellular superoxide dismutase (EC-SOD) or with the EC-SOD gene knockout (KO). Adult EC-SOD mice and their wild type littermates and EC-SOD KO mice and their wild type littermates were exposed to 100% oxygen for 6 hours and for 72 hours, and the hearts were subsequently removed under …

Chemical Evidence For Dietary Toxin Sequestration In The Asian Snake Rhabdophis Tigrinus, Deborah A. Hutchinson

Biological Sciences Theses & Dissertations

Rhabdophis tigrinus (Colubridae: Natricinae) is an oviparous, bufophagous (toad-eating) snake from eastern Asia that possesses defensive integumentary glands on the neck known as nuchal glands. These glands are used in antipredator displays and typically contain bufadienolide toxins. Whereas toads are known to synthesize bufadienolide steroids from cholesterol precursors, we found that chemically undefended R. tigrinus must sequester bufadienolides from ingested toads in order to exhibit these compounds in their nuchal glands. Chemically defended females are capable of provisioning their embryos with these toxins so their unfed hatchlings possess defensive bufadienolides prior to consuming toads themselves. All of the hatchling R. …

Model Studies Towards The Total Synthesis Of Lyconadin A Via An Acyl Radical Cascade Reaction, Koudi Zhu

Theses and Dissertations

Lyconadin A is an alkaloid possessing a unique structure and antitumor activity. The total synthesis of Lyconadin A was proposed via an acyl radical cascade reaction. To investigate the possibility and stereoselectivity of the cascade cyclization, phenyl selenoester 16 was chosen as a model substrate to study the 7-exo-5-exo radical cyclization. A synthetic route to phenyl selenoester 16 was developed. The 7-exo-5-exo radical cyclization was found to occur with a high yield and excellent stereoselectivty. Attempts were also tried to synthesize another radical precursor 14 albeit with less success. A synthetic pathway to the synthesis of 14 as well as …

Selenoproteins And Selenoproteomes, Vadim N. Gladyshev

Vadim Gladyshev Publications

In the past several years, progress in genome sequencing and development of specialized bioinformatics tools allowed efficient identification of selenocysteine-containing proteins encoded in completely sequenced genomes. Information is currently available on selenoproteomes from a variety of organisms, including humans, which contain 25 known selenoprotein genes. This review provides basic information about mammalian selenoproteins and other known selenoprotein families. Analysis of full sets of selenoproteins in organisms provides exciting avenues for examining selenoprotein evolution and dependence of organisms on the trace element selenium and allows linking selenoproteins with specific biological and biomedical effects of dietary selenium.

Mammalian And Other Eukaryotic Selenocysteine Trnas, Bradley A. Carlson, Xue-Ming Xu, Rajeev Shrimali, Aniruddha Sengupta, Min-Hyuk Yoo, Robert Irons, Nianxin Zhong, Dolph L. Hatfield, Byeong Jae Lee, Alexei V. Lobanov, Vadim N. Gladyshev

Vadim Gladyshev Publications

Selenocysteine (Sec) tRNA occupies a prominent position in the expression of selenoproteins as it is essential for their synthesis and it provides the means by which selenium is co-translationally inserted into protein as the amino acid, Sec. Thus, Sec tRNA is regarded as the principle constituent in selenoprotein synthesis. Many features unique to this tRNA have been characterized over the years in mammals and other eukaryotes. In the last five years, the major advances have been in an elucidation of the different roles that the two major Sec tRNA isoforms play in selenoprotein biosynthesis and in Sec biosynthesis. One isoform …

"Preface" To Selenium: Its Molecular Biology And Role In Human Health, Second Edition, Dolph L. Hatfield, Marla J. Berry, Vadim N. Gladyshev

Vadim Gladyshev Publications

The purpose of the new edition book is to inform the reader of many new discoveries and to examine our present knowledge of the molecular biology of selenium, its incorporation into proteins as selenocysteine and the role that this element and selenium-containing proteins (selenoproteins) play in health. The book's emphasis is on our understanding of selenium metabolism in mammals and the role of this element in human health. The book begins with a brief history of selenium and how its face has changed through the years fiom one of a toxin and possible carcinogen to one of an essential micronutrient …

Mouse Models For Assessing The Role Of Selenoproteins In Health And Development, Bradley A. Carlson, Xue-Ming Xu, Rajeev Shrimali, Aniruddha Sengupta, Min-Hyuk Yoo, Nianxin Zhong, Dolph L. Hatfield, Robert Irons, Cindy D. Davis, Byeong Jae Lee, Sergey V. Novoselov, Vadim N. Gladyshev

Vadim Gladyshev Publications

Mouse models have been generated to assess the roles of selenoproteins involved with housekeeping tasks and/or stress-related phenomena in development and health. Each mouse model has taken advantage of the fact that the synthesis of all selenoproteins is dependent on the expression of two selenocysteine ^{(Sec) tRNA[Ser]Sec} isoforms that differ fiom each other by a single methyl group on the ribosyl moiety at position 34. The endogenous ^{(Sec) tRNA[Ser]Sec} population was selectively altered by generating mouse models involving 1) transgenic animals carrying mutant or wild type ^{(Sec) tRNA[Ser]Sec} transgenes, 2) conditional knockout animals carrying a floxed ^{(Sec) tRNA[Ser]Sec} gene that …

Selenium And Methionine Sulfoxide Reduction, Hwa-Young Kim, Vadim N. Gladyshev

Vadim Gladyshev Publications

Methionine residues in proteins can be readily oxidized to a diastereomeric mixture of methionine sulfoxides by reactive oxygen species. In most organisms, methionine sulfoxides are reversibly and stereospecifically reduced back to methionine by two distinct classes of repair enzymes, methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). Methionine sulfoxide reduction is thought to be an essential pathway that protects cells from oxidative stress and regulates protein function. This pathway is also implicated in delaying the aging process in organisms from yeast to mammals. The first selenoprotein identified using bioinformatics methods, SelR (also known as SelX or MsrB1), was recently found to …

Evolution Of Selenocysteine Decoding And The Key Role Of Selenophosphate Synthetase In The Pathway Of Selenium Utilization, Gustavo Salinas, Héctor Romero, Xue-Ming Xu, Bradley A. Carlson, Dolph L. Hatfield, Vadim N. Gladyshev

Vadim Gladyshev Publications

The complete sequencing of genomes and the development of in silico methods for identification of genes encoding selenocysteine (Sec)-containing proteins have greatly contributed to shape our view on the evolution of selenium utilization in nature. Current evidence is consistent with the idea that Sec decoding is a late addition to the genetic code and it evolved once, before the separation of archaeal, bacterial and eukaryal domains. Many organisms have lost the Sec decoding trait, but recent evidence has shown that the loss is not irreversible. The distribution of organisms that use UGA as a Sec codon suggests that Sec decoding …

Selenoproteins In Parasites, Gustavo Salinas, Alexei V. Lobanov, Vadim N. Gladyshev

Vadim Gladyshev Publications

Parasites, which cause an enormous burden in the population of the third world, are a diverse group of organisms, many of which are sensitive to oxidative stress imposed by their hosts. In recent years, several selenoprotein families, some with antioxidant properties, have been described and characterized in metazoan parasites. Glutathione peroxidase and thioredoxin glutathione reductase (TGR) appear to be essential selenoproteins in flatworms (phylum Platyhelminthes). TGR is the single enzyme that provides reducing equivalents to both thioredoxin and glutathione pathways, in contrast to hosts, which evolve parallel pathways. In roundworms (phylum Nematoda), selenoproteins have recently been described, revealing …

The 15-Kda Selenoprotein (Sep15): Functional Analysis And Role In Cancer, Vyacheslav M. Labunskyy, Vadim N. Gladyshev, Dolph L. Hatfield

Vadim Gladyshev Publications

The 15-kDa selenoprotein (Sep15) was identified several years ago as a protein of unknown function. In recent years, several lines of evidence implicated Sepl5 in the effect of dietary selenium in cancer prevention. These lines of evidence include: 1) protein expression patterns in normal and malignant cells; 2) identification of polymorphic sites that regulate Sep15 levels and differentially respond to selenium supplementation; 3) location of the Sep15 gene in the human genome; and 4) correlation between Sep15 haplotype and susceptibility to cancer. Functional analyses revealed a specific interaction between Sep15 and a protein folding sensor in the endoplasmic reticulum of …

The Mechanism Of Assembly Of The G-Protein Beta Gamma Subunit Dimer By Ck2 Phosphorylated Phosducin-Like Protein And The Chaperonin Containing Tcp-1, Christine M. Baker

Theses and Dissertations

Phosducin-like protein (PhLP) binds G-protein beta gamma subunits and is thought to assist in assembly of the G-protein beta gamma dimer. Phosphorylation of PhLP at serine residues 18-20 by the casein kinase 2 (CK2) appears to play an essential role in this process. PhLP has also been shown to interact with the chaperonin containing TCP-1 (CCT) atop its apical domain, not entering the substrate folding cavity. However, the physiological role of the PhLP-CCT interaction in G-protein beta gamma dimer formation remains unclear. This study addresses the mechanism of G-protein beta gamma assembly by exploring the specific roles of CCT and …

Fabrication Of Polymeric Microfluidic Devices For Protein Analysis, Jikun Liu

Theses and Dissertations

2-Bromoisobutyryl bromide was immobilized on poly(methyl methacrylate) (PMMA) substrates activated using an oxygen plasma. Atom-transfer radical polymerization was then performed to graft poly(ethylene glycol) (PEG) on the PMMA surface. PMMA micro capillary electrophoresis (µCE) devices made with the covalently modified surfaces exhibited substantially reduced electroosmotic flow and nonspecific adsorption of proteins. Both column efficiency and migration time reproducibility were one order of magnitude better with derivatized PMMA µCE devices compared to untreated versions. Fast, reproducible, and efficient separations of proteins and peptides were demonstrated using the PEG-grafted PMMA µCE chips. All analyses were completed in less than 60 seconds, and …

(Review) Green Fluorescent Proteins, Marc Zimmer

Chemistry Faculty Publications

Reviews the book:Green Fluorescent Protein: Properties, Applications, and Protocols. Second Edition. Methods of Biochemical Analysis, Volume 47. Edited by Martin Chalfie and Steven R Kain.Green Fluorescent Protein: Properties, Applications, and Protocols. Second Edition. Methods of Biochemical Analysis, Volume 47. Edited by Martin Chalfie and Steven R Kain. Hoboken (New Jersey): Wiley-Interscience. $89.95. xv + 443 p + 24 pl; ill.; index. ISBN: 0–471–73682–1. 2006.

Some Statistical Issues In Microarray Gene Expression Data, Matthew S. Mayo, Byron J. Gajewski, Jeffrey S. Morris

Jeffrey S. Morris

In this paper we discuss some of the statistical issues that should be considered when conducting experiments involving microarray gene expression data. We discuss statistical issues related to preprocessing the data as well as the analysis of the data. Analysis of the data is discussed in three contexts: class comparison, class prediction and class discovery. We also review the methods used in two studies that are using microarray gene expression to assess the effect of exposure to radiofrequency (RF) fields on gene expression. Our intent is to provide a guide for radiation researchers when conducting studies involving microarray gene expression …

Molecular Validation Of Lpxc As An Antibacterial Drug Target In Pseudomonas Aeruginosa, Adam Barb, Khisimuzi E. Mdluli, Pamela R. Witte, Toni Kline, Alice L. Erwin, Bryce E. Mansfield, Amanda L. Mcclerren, Michael C. Pirrung, L. Nathan Tumey, Paul Warrener, Christian R. H. Raetz, C. Kenall Stover

Adam Barb

LpxC [UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase] is a metalloamidase that catalyzes the first committed step in the biosynthesis of the lipid A component of lipopolysaccharide. A previous study (H. R. Onishi, B. A. Pelak, L. S. Gerckens, L. L. Silver, F. M. Kahan, M. H. Chen, A. A. Patchett, S. M. Galloway, S. A. Hyland, M. S. Anderson, and C. R. H. Raetz, Science 274:980-982, 1996) identified a series of synthetic LpxC-inhibitory molecules that were bactericidal for Escherichia coli. These molecules did not inhibit the growth of Pseudomonas aeruginosa and were therefore not developed further as antibacterial drugs. The inactivity of the LpxC …

Hyperhomocysteinemia And Coronary Artery Disease In Pakistan, M Perwaiz Iqbal

Department of Biological & Biomedical Sciences

The relative risk of developing coronary artery disease (CAD) in Pakistani men is highest in early ages. Majority of those suffering from CAD belong to the lower middle socioeconomic stratum of the society. Mild hyperhomocysteinemia (concentration of plasma homocysteine between 15-25 micromol/l) is very commonly seen in Pakistani patients with acute myocardial infarction (AMI) as well as in normal healthy subjects. There appears to be a lack of association between hyperhomocysteinemia and CAD in Pakistani population. There is also no evidence of association of methylenetetrahydrofolate reductase 677C>T mutation with CAD in this population. High prevalence of deficiency of folate …

Membrane Organization And Multimeric Interactions Of The Aer Receptor In E. Coli, Divya Dashrathkumar Amin

Loma Linda University Electronic Theses, Dissertations & Projects

Escherichia coli swims toward niches ideal for generating maximum energy by monitoring the environment with the aerotaxis sensor, Aer. This receptor has an N-terminal PAS domain, which is a sensor module present in a variety of proteins from all kingdoms of life (including humans). To date, the mechanism of signaling from a PAS sensor domain to a single signaling receiver domain has not been resolved. In Aer, the PAS sensor is separated from the signaling region by a membrane anchor. Most "transmembrane prediction" programs forecast a single membrane span for this membrane anchor, but such a topology is inconsistent our …

Recruitment Of Bad By The Chlamydia Trachomatis Vacuole Correlates With Host-Cell Survival, Philippe Verbeke, Lynn Welter-Stahl, S. Ying, J. Hansen, Georg Hacker, Toni Darville, David M. Ojcius

All Dugoni School of Dentistry Faculty Articles

Chlamydiae replicate intracellularly in a vacuole called an inclusion. Chlamydial-infected host cells are protected from mitochondrion-dependent apoptosis, partly due to degradation of BH3-only proteins. The host-cell adapter protein 14-3-3β can interact with host-cell apoptotic signaling pathways in a phosphorylation-dependent manner. In Chlamydia trachomatis-infected cells, 14-3-3β co-localizes to the inclusion via direct interaction with a C. trachomatis-encoded inclusion membrane protein. We therefore explored the possibility that the phosphatidylinositol-3 kinase (PI3K) pathway may contribute to resistance of infected cells to apoptosis. We found that inhibition of PI3K renders C. trachomatis-infected cells sensitive to staurosporine-induced apoptosis, which is accompanied by mitochondrial cytochrome c …

Desulfovibrio Desulfuricans G20 Tetraheme Cytochrome Structure At 1.5 A˚ And Cytochrome Interaction With Metal Complexes, Mrunalini Pattarkine, J J. Tanner, C A. Bottoms, Y H. Lee, Judy D. Wall

Faculty Works

The structure of the type I tetraheme cytochrome c3 from Desulfovibrio desulfuricans G20 was determined to 1.5 A˚ by X-ray crystallography. In addition to the oxidized form, the structure of the molybdate-bound form of the protein was determined from oxidized crystals soaked in sodium molybdate. Only small structural shifts were obtained with metal binding, consistent with the remarkable structural stability of this protein. In vitro experiments with pure cytochrome showed that molybdate could oxidize the reduced cytochrome, although not as rapidly as U(VI) present as uranyl acetate. Alterations in the overall conformation and thermostability of the metal-oxidized protein were investigated …

Thioredoxin Reductase 1 Deficiency Reverses Tumor Phenotype And Tumorigenicity Of Lung Carcinoma Cells, Min-Hyuk Yoo, Xue-Ming Xu, Bradley A. Carlson, Vadim N. Gladyshev, Dolph L. Hatfield

Vadim Gladyshev Publications

Dietary selenium has potent cancer prevention activity. Both low molecular weight selenocompounds and selenoproteins are implicated in this effect. Thioredoxin reductase 1 (TR1) is one of the major antioxidant and redox regulators in mammals that supports p53 function and other tumor suppressor activities. However, this selenium-containing oxidoreductase is also overexpressed in many malignant cells and has been proposed as a target for cancer therapy. To further assess the role of TR1 in the malignancy process, we used RNA interference technology to decrease its expression in mouse lung carcinoma (LLC1) cells. Stable transfection of LLC1 cells with a small interfering RNA …

In Vitro Expression And Purification Of Class I Mhc Molecules, Loi Cheng

Honors Scholar Theses

The major histocompatibility complex (MHC) is a gene family responsible for many critical functions of the immune system in most vertebrates. The MHC consists of three classes differentiated by their structure and function, and MHC class I encodes antigen binding proteins as well as chaperone and accessory proteins such as tapasin. The purpose of this project is to reconstitute several human MHC class I molecules in their peptide-filled and peptide-deficient forms, and to purify these proteins for biochemical study. The expressed proteins include wild type and mutant variants of the fusion protein human leukocyte antigen HLA-B*0801-fos, and human beta-2-microglobulin (β2m). …

Vitronectin And Plasminogen Activator Inhibitor-1 Form Higher-Order Complexes That Localize To The Extracellular Matrix And Adopt Adhesive Properties, Kenneth H. Minor

Doctoral Dissertations

Vitronectin is a human circulatory protein that is recruited to the extracellular matrix (ECM) during tissue remodeling pathways linked to injury, inflammation, and tumor metastasis. Whereas circulating vitronectin is monomeric, the tissue-associated form is multimeric and contains multivalent binding sites for both cell-surface receptors and components of the extracellular matrix. In addition, matrix-associated vitronectin directly regulates plasmin-regulated matrix proteolysis through the localization and stabilization of the serine protease inhibitor, plasminogen activator inhibitor type-1 (PAI-1). As a component of the ECM, vitronectin adopts a pro-adhesive role, providing a unique regulatory link between cell adhesion and pericellular proteolysis. The mechanism by which …

Probability Of Real -Time Detection Vs Probability Of Infection For Aerosolized Biowarfare Agents: A Model Study., Alexander G. Sabelnikov, Vladimir Zhukov, C Ruth Kempf

Alexander G Sabelnikov

No abstract provided.

Evaluation Of Toxicity Following Electrically Mediated Interleukin-12 Gene Delivery In A B16 Mouse Melanoma Model, Loree Heller, Kathleen Merkler, Jeffrey Westover, Yolmari Cruz, Domenico Coppola, Kaaron Benson, Adil Daud, Richard Heller

Bioelectrics Publications

PURPOSE: Interleukin-12 (IL-12) has potential as an immunotherapeutic agent for the treatment of cancer but is unfortunately associated with toxicity. Delivery of a plasmid encoding IL-12 with electroporation induces an antitumor effect in the B16 mouse melanoma model without serious side effects. To translate this observation to the clinic, an evaluation of toxicity was done in the mouse model.

EXPERIMENTAL DESIGN: Weight change, tumor response, blood chemistry and hematology values, and serum IL-12 levels were evaluated. Multiple tissues were analyzed histopathologically.

RESULTS: A pronounced reduction in tumor volume, including a large percentage of complete regressions, was observed after electrically mediated …

Isolation Of A Ubiquitous Obligate Thermoacidophilic Archaeon From Deep-Sea Hydrothermal Vents, Anna-Louise Reysenbach, Yitai Liu, Amy B. Banta, Terry J. Beveridge, Julie D. Kirshtein, Stefan Schouten, Margaret K. Tivey, Karen L. Von Damm, Mary A. Voytek

Biology Faculty Publications and Presentations

Deep-sea hydrothermal vents are important in global biogeochemical cycles, providing biological oases at the sea floor that are supported by the thermal and chemical flux from the Earth's interior. As hot, acidic and reduced hydrothermal fluids mix with cold, alkaline and oxygenated sea water, minerals precipitate to form porous sulphide–sulphate deposits. These structures provide microhabitats for a diversity of prokaryotes that exploit the geochemical and physical gradients in this dynamic ecosystem. It has been proposed that fluid pH in the actively venting sulphide structures is generally low (pH < 4.5), yet no extreme thermoacidophile has been isolated from vent deposits. Culture-independent surveys based on ribosomal RNA genes from deep-sea hydrothermal deposits have identified a widespread euryarchaeotal lineage, DHVE2 (deep-sea hydrothermal vent euryarchaeotic 2) Despite the ubiquity and apparent deep-sea endemism of DHVE2, cultivation of this group has been unsuccessful and thus its metabolism remains a mystery. Here we report the isolation and cultivation of a member of the DHVE2 group, which is an obligate thermoacidophilic sulphur- or iron-reducing heterotroph capable of growing from pH 3.3 to 5.8 and between 55 and 75 °C. In addition, we demonstrate that this isolate constitutes up to 15% of the archaeal population, providing evidence that thermoacidophiles may be key players in the sulphur and iron cycling at deep-sea vents.

Structure-Function Analysis Of Nrage: A Protein Involved In Developmental Neural Apoptosis, Rebecca Cowling

Electronic Theses and Dissertations

The Neurotrophins and Bone Morphogenic Proteins (BMP) have both been implicated in the maintenance of cellular proliferation and apoptosis in the developing nervous system. Downstream of both signaling pathways is NRAGE, a member of the Melanoma Antigen (MAGE) gene family and, under BMP stimulation, activates p38 leading to caspase 3 cleavage. NRAGE possesses a highly conserved MAGE Homology Domain (MHD) and a second, less well conserved MHD (denoted MHD2) as well as a unique 25 tandem WQXPXX hexapeptide repeat region. Binding partners for the MHD and hexapeptide repeat regions have been identified but their cellular consequences have not been defined. …

The Fate Of Iron Released From Heme By Hemeoxygenase-1, Jonathan Mark Gardner

Undergraduate Honors Capstone Projects

A strain of Escherichia coli was genetically modified to co-express human heme oxygenase-1 and ferritin. The E. coli were then grown with varying amounts of hemin to see if the iron released upon degradation of the hemin by heme oxygenase-1 is loaded into ferritin. Following incubation, the ferritin was purified and the amount of iron loaded into ferritin determined. It was found that ferritin purifed from E. coli expressing human heme oxygenase-1 contained more iron than E. coli that did not contain human heme oxygenase-1. It was concluded that some of the iron released upon degradation of hemin by heme …

Mechanisms Imposing The Vbeta Bias Of V14a Natural Killer T Cells And Consequences For Microbial Glycolipid Recognition, Paul B. Savage, Dasten G. Wei, Shane A. Curran, Luc Teyton, Albert Bendelac

Faculty Publications

Mouse and human natural killer T (NKT) cells recognize a restricted set of glycosphingolipids presented by CD1d molecules, including self iGb3 and microbial alpha-glycuronosylceramides. The importance of the canonical Valpha 14-Jalpha18 TCR alphachain for antigen recognition by NKT cells is well recognized, but the mechanisms underlying the Vbeta8, Vbeta7, and Vbeta2 bias in mouse have not been explored. To study the influences of thymic selection and the constraints of pairing with Valpha 14-Jalpha 18, we have created a population of mature T cells expressing Valpha 14-Jalpha 18 TCRalpha chain in CD1d-deficient mice and studied its recognition properties in vitro and …