Biochemistry, Biophysics, and Structural Biology Commons^™

The Role Of A Conserved Serine Residue Within Hydrogen Bonding Distance Of Fad In Redox Properties And The Modulation Of Catalysis By Ca2+/Calmodulin Of Constitutive Nitric-Oxide Synthases, Satya Prakash Panda, Ying Tong Gao, Linda J. Roman, Pavel Marta´Sek, John C. Salerno, Bettie Masters

Faculty Articles

The crystal structure of the neuronal nitric-oxide synthase (nNOS) NADPH/FAD binding domain indicated that Ser-1176 is within hydrogen bonding distance of Asp-1393 and the O4 atom of FAD and is also near the N5 atom of FAD (3.7Å). This serine residue is conserved in most of the ferredoxin-NADP⁺ reductase family of proteins and is important in electron transfer. In the present study, the homologous serines of both nNOS (Ser-1176) and endothelial nitric-oxide synthase (eNOS) (Ser-942) were mutated to threonine and alanine. Both substitutions yielded proteins that exhibited decreased rates of electron transfer through the flavin domains, in the presence …

The Structure And Function Of Frataxin, Krisztina Z. Bencze, Kalyan C. Kondapalli, Jeremy D. Cook, Stephen Mcmahon, César Millán-Pacheco, Nina Pastor, Timothy L. Stemmler

Biochemistry and Molecular Biology Faculty Publications

Frataxin, a highly conserved protein found in prokaryotes and eukaryotes, is required for efficient regulation of cellular iron homeostasis. Humans with a frataxin deficiency have the cardio- and neurodegenerative disorder Friedreich’s ataxia, commonly resulting from a GAA trinucleotide repeat expansion in the frataxin gene. While frataxin’s specific function remains a point of controversy, a general consensus is the protein assists in controlling cellular iron homeostasis by directly binding iron. This review focuses on the structural and biochemical aspects of iron binding by the frataxin orthologs and outlines molecular attributes that may help explain the protein’s role in different cellular pathways.

The Effects Of Ultrashort Pulsed Electric Fields On The Fluorescent Molecule Bodipy Fl C5 Ceramide, Rachael Sara Shevin

Electrical & Computer Engineering Theses & Dissertations

Nanosecond pulsed electric fields are able to manipulate internal cell organelles by taking advantage of the small but finite time required for charge to establish across a capacitive barrier. Pulsed electric fields also affect macromolecular structures such as enzymes and proteins. Application of a single nanosecond pulsed electric field decreases the fluorescence of a commercially available biological dye Bodipy FL C₅ Ceramide, used to observe the Golgi apparatus of living cells. The effect is seen when the fluorophore is exposed to an electric field both in vitro and when incorporated into the Golgi of a living Jurkat cell. Observation …

Erv26p Directs Pro-Alkaline Phosphatase Into Endoplasmic Reticulum–Derived Coat Protein Complex Ii Transport Vesicles, Catherine A. Bue, Christine M. Bentivoglio, Charles Barlowe

Dartmouth Scholarship

Secretory proteins are exported from the endoplasmic reticulum (ER) in transport vesicles formed by the coat protein complex II (COPII). We detected Erv26p as an integral membrane protein that was efficiently packaged into COPII vesicles and cycled between the ER and Golgi compartments. The erv26Δ mutant displayed a selective secretory defect in which the pro-form of vacuolar alkaline phosphatase (pro-ALP) accumulated in the ER, whereas other secretory proteins were transported at wild-type rates. In vitro budding experiments demonstrated that Erv26p was directly required for packaging of pro-ALP into COPII vesicles. Moreover, Erv26p was detected in a specific complex with pro-ALP …

Agswe1p Regulates Mitosis In Response To Morphogenesis And Nutrients In Multinucleated Ashbya Gossypii Cells, Hanspeter Helfer, Amy S. Gladfelter

Dartmouth Scholarship

Nuclei in the filamentous, multinucleated fungus Ashbya gossypii divide asynchronously. We have investigated what internal and external signals spatially direct mitosis within these hyphal cells. Mitoses are most common near cortical septin rings found at growing tips and branchpoints. In septin mutants, mitoses are no longer concentrated at branchpoints, suggesting that the septin rings function to locally promote mitosis near new branches. Similarly, cells lacking AgSwe1p kinase (a Wee1 homologue), AgHsl1p (a Nim1-related kinase), and AgMih1p phosphatase (the Cdc25 homologue that likely counteracts AgSwe1p activity) also have mitoses distributed randomly in the hyphae as opposed to at branchpoints. Surprisingly, however, …

Cell Edge Features Affected By Microtubule Inhibitor Combinations, Sonal Uppal, Nancy Boudreau, Elizabeth Wendt, Carol Heckman

Applied Statistics and Operations Research Faculty Publications

The tumor promoter, phorbol 12-myristate 13-acetate (PMA), enhances tumor yield through an epigenetic mechanism. PMA, like another promoter, phosphatase inhibitor okadaic acid, works by maintaining proteins in a phosphorylated state. In order to identify chemicals with promoter and antipromoter effects, this laboratory has developed a standard curve of morphogenetic changes using data from precancerous cell lines that eventually became neoplastic. Using the curve as a basis of comparison, we defined the “signature” phenotype as that adopted when a cell line became neoplastic. The results of solving for signature type disclosed that the microtubule-depolymerizing compound, colchicine, had a promoter-like effect [2]. …

Myod Synergizes With The E-Protein Heb Beta To Induce Myogenic Differentiation, Maura H. Parker, Robert L.S. Perry, Melanie C. Fauteux, Charlotte A. Berkes, Michael A. Rudnicki

Biology Faculty Publications

The MyoD family of basic helix-loop-helix transcription factors function as heterodimers with members of the E-protein family to induce myogenic gene activation. The E-protein HEB is alternatively spliced to generate alpha and beta isoforms. While the function of these molecules has been studied in other cell types, questions persist regarding the molecular functions of HEB proteins in skeletal muscle. Our data demonstrate that HEB alpha expression remains unchanged in both myoblasts and myotubes, whereas HEB beta is upregulated during the early phases of terminal differentiation. Upon induction of differentiation, a MyoD-HEB beta complex bound the E1 E-box of the myogenin …

Development Of An In Vitro Protocol To Study The Effect Of Estrogen On Osteoblast Activity, Sourik Ganguly

Morehead State Theses and Dissertations

A thesis presented to the faculty of the College of Science & Technology at Morehead State University in partial fulfillment of the requirements for the Degree of Master of Science by Sourik Ganguly on July 7, 2006.

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 …

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 …

Cloning And Overexpression Of Imp/Osta, An Essential Escherichia Coli Outer Membrane Protein With A Possible Role In Lipid Transport, Leslie Lekatz

Honors Capstones

Capstone submitted as a graduation requirement for the BSU Honors Program.

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 …

Nanosecond Pulsed Electric Field Effects On Cell Cycle And Apoptosis, Emily H. Hall

Theses and Dissertations in Biomedical Sciences

Apoptosis, programmed cell death, is a highly regulated and complex pathway essential for embryonic development, immune-system function and maintenance of tissue homeostasis where cells induce their own cell death. Cells undergoing apoptosis exhibit a distinctive phenotype characterized by maintenance of membrane integrity, cell shrinkage, phosphatidylserine (PS) externalization at the plasma membrane, caspase protease activation, DNA fragmentation, release of cytochrome c from the mitochondrion, and membrane blebbing. An important regulatory protein in the apoptotic pathway is p53. The p53 protein functions to modulate the cell cycle by arresting cells in the G1 and G 2 phases to repair DNA damage, and/or …

The Study Of Nitric Oxide Synthase Expression, Function, And Regulation In The Renal Vasculature During Postnatal Renal Development, Brian Blake Ratliff

Theses and Dissertations in Biomedical Sciences

The newborn kidney is vulnerable to vasomotor acute renal failure (ARF) from adverse perinatal events or complications of prematurity. Nitric oxide (NO) vasodilation is vitally protective in this type of ARF, but its relationship with other vasoactive factors, such as angiotensin II (AII) has not been examined. In the immature kidney, nitric oxide synthase (NOS) isoforms, specifically eNOS and nNOS, are developmentally regulated, but their specific role and regulation are unknown.

The enhanced vasodilatory role of NO in the immature kidney was hypothesized to be attributed to regulatory, expressional, and functional differences in eNOS and nNOS isoforms from the adult. …

Modulation Of Tgfβ-Induced Pai -1 Expression By Changes In Actin Polymerization In Human Mesangial Cells, Keyur Patel

Theses and Dissertations in Biomedical Sciences

Chronic renal diseases show increased deposition of extracellular matrix (ECM) in the glomerulus (glomerulosclerosis). Glomerulosclerosis is associated with activation of normally quiescent glomerular mesangial cells into myofibroblast-like cells. The overall objective of this study is to delineate cellular mechanism/s of myofibroblast-differentiation in disease states. In cultured mesangial cells certain characteristics of myofibroblast differentiation (α-smooth muscle actin (α-SMA) and hypertrophy) are associated with an increase in polymeric actin microfilaments (stress fibers). It is likely that other genes are also regulated in an actin cytoskeleton-dependent manner during myofibroblast differentiation. In these studies, we therefore examined the hypothesis that changes in the actin …

Aspartyl-Trna Synthetase Is The Target Of Peptidenucleotide Antibiotic Microcin C, Anastasia Metlitskaya, Teymur Kazakov, Aigar Kommer, Olga Pavlova, Mette Praetorius-Ibba, Michael Ibba, Igor Krasheninnkov, Vyacheslav Kolb, Inessa Khmel, Konstantin Severinov

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Microcin C is a ribosome-synthesized heptapeptide that contains a modified adenosine monophosphate covalently attached to the C-terminal aspartate. Microcin C is a potent inhibitor of bacterial cell growth. Based on the in vivo kinetics of inhibition of macromolecular synthesis, Microcin C targets translation, through a mechanism that remained undefined. Here, we show that Microcin C is a subject of specific degradation inside the sensitive cell. The product of degradation, a modified aspartyl-adenylate containing an N-acylphosphoramidate linkage, strongly inhibits translation by blocking the function of aspartyl-tRNA synthetase.

Three-Dimensional Structure Of The Bacterial Cell Wall Peptidoglycan, Samy O. Meroueh, Krisztina Z. Bencze, Dusan Hesek, Mijoon Lee, Timothy L. Stemmler, Shahriar Mobashery

Biochemistry and Molecular Biology Faculty Publications

The 3D structure of the bacterial peptidoglycan, the major constit- uent of the cell wall, is one of the most important, yet still unsolved, structural problems in biochemistry. The peptidoglycan comprises alternating N-acetylglucosamine (NAG) and N-acetylmu- ramic disaccharide (NAM) saccharides, the latter of which has a peptide stem. Adjacent peptide stems are cross-linked by the transpeptidase enzymes of cell wall biosynthesis to provide the cell wall polymer with the structural integrity required by the bacte- rium. The cell wall and its biosynthetic enzymes are targets of antibiotics. The 3D structure of the cell wall has been elusive because of its …

C To U Editing Stimulates A To I Editing In The Anticodon Loop Of A Cytoplasmic Threonyl Trna In Trypanosoma Brucei, Mary Anne T. Rubio, Frank L. Ragone, Kirk W. Gaston, Michael Ibba, Juan D. Alfonzo

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Editing of tRNAs is widespread in nature and either changes the decoding properties or restores the folding of a tRNA. Unlike the phylogenetically disperse adenosine (A) to inosine (I) editing, cytosine (C) to uridine (U) editing has only been previously described in organellar tRNAs. We have shown that cytoplasmic tRNA^Thr(AGU) undergoes two distinct editing events in the anticodon loop: C to U and A to I. In vivo, every inosine-containing tRNA^Thr is also C to U edited at position 32. In vitro, C to U editing stimulates conversion of A to I at the wobble base. Although …

Use Of Cerium Oxide Nanoparticles For Protection Against Radiation-Induced Cell Death, Jimmie Colon

Electronic Theses and Dissertations

The ability of engineered cerium oxide nanoparticles to confer radioprotection was examined. Rat astrocytes were treated with cerium oxide nanoparticles to a final concentration of 10 nanomolar, irradiated with a single 10 Gy dose of ionizing radiation and cell death was evaluated by propidium iodine uptake at 24 and 48 hours after radiation insult. Treatment of rat astrocytes with nanoceria resulted in an approximate 3-fold decrease in radiation induced death. These results suggest that the nanoceria are conferring protection from radiation induced cell death. Further experiments with human cells were conducted. Human normal and tumor cells (MCF-7 and CRL8798) were …

Characterization And Evaluation Of The Immunogenizity Of Chloroplast-Derived 19-Kilodalton C-Terminal Merozoite Surface Antigen 1 (Msp1) Of Plasmodium Yoelii Yoelii, Sushamadevi Kamarajugadda

Electronic Theses and Dissertations

Malaria is a protozoan disease caused in humans by four different species of the genus Plasmodium (P. falciparum, P. vivax, P. ovale, P. malarie) and in rodents by Plasmodium yoelii yoelii. It has been reported that 1.5 to 3 million deaths occur worldwide due to malaria and the DALY (Daily affected life years) reports about 0.76% of world population affected by the disease in some of the major countries like Africa, Asia, Latin America etc., Due to the development of resistance to drugs by the parasite, there is an urgent need and prime importance for the development of an effective …

Expression Of Heterologous Proteins In Transgenic Tobacco Chloroplasts To Produce A Biopharmaceutical And Biopolymer, Andrew Leon Devine

Electronic Theses and Dissertations

The chloroplast has been demonstrated to be an ideal compartment to accumulate certain proteins or their biosynthetic products that would be harmful if they were accumulated in the cytoplasm. Hyper-expression of foreign proteins in chloroplast transgenics has accumulated up to 46% total soluble protein, this is possible due to the ~100 chloroplast genomes per chloroplast and ~100 chloroplasts per cell which can therefore, contain up to 10,000 copies of the transgene. Maternal gene inheritance of plastids in most crop plants results in natural gene containment. Chloroplast transformation also eliminates positional effects that are frequently observed with nuclear transformation and no …

Epichlorohydrin Cross-Linking Of Synthetic Dna Oligomers, Rami W. Zaharan, Julie T. Millard

Undergraduate Research Symposium (UGRS)

Epichlorohydrin (ECH), an important chemical in the synthetic polymer industry, is a bifunctional alkylating agent with the potential to form DNA interstrand crosslinks. Occupational exposure to this suspect carcinogen leads to chromosomal aberrations, and ECH has been shown to undergo reaction with DNA in vivo and in vitro. We are using denaturing polyacrylamide gel electrophoresis to assess cross-linking of synthetic DNA oligomers by both ECH and the related compound, epibromohydrin (EBH). Both epihalohydrins produce a low-mobility band on denaturing gels consistent with an interstrand cross-link. Moreover, the efficiencies, sequence preferences, reaction kinetics, and pH dependence differ for the two compounds, …

Regulation Of Apoptotic Alkalinization Through Phosphorylation Of Sodium Hydrogen Exchanger Via P38 Mitogen Activated Protein Kinase, Amy Greinier

Electronic Theses and Dissertations

Regulation of intracellular pH is responsible for many cellular processes, such as metabolism, cell cycle progression, and apoptosis. Many chemotherapeutic agents work by inducing target cells to undergo apoptosis, a cell death process still poorly understood. Previous studies demonstrated that a rise in intracellular pH activated apoptotic proteins leading to cytochrome C release. This "apoptotic alkalinization" occurred upon activation of the plasma membrane protein, sodium hydrogen exchanger-1 (NHE1), whose activity is regulated by the stress kinase p38 MAPK. In previous studies, upon cytokine withdrawal from cytokine-dependent lymphocytes induced the activity of the p38 MAP kinase which then phosphorylated the C-terminus …

Expression And Characterization Of Mycobacterium Paratuberculosis 19kda With Posttranslational Modification, Mitra Safavi-Khasraghi

Electronic Theses and Dissertations

Despite the fact that E. coli supports limited posttranslational modification, this bacterium has been universally used as the expression system of choice. Expression of modified proteins in E. coli may lead to expression of recombinant proteins that lack essential immunomodulatory or catalytic components essentials for infectious processes. Previously in our laboratory, pMptb#28 plasmid containing a 4.8 kb insert from M. paratuberculosis has been identified which expressed 16 kDa recombinant protein in E. coli and 19 kDa recombinant protein in Mycobacterium smegmatis. The objective of this study is to identify the ORF sequence, investigate possible posttranslational modification and characterize the protein …

Bioinformatic Analysis Of Solanaceae Chloroplast Genomes And Characterization Of An Arabidopsis Protein Disulfide Isomerase In Transgenic Tobacco Chloroplasts, Justin James Grevich

Electronic Theses and Dissertations

Throughout history, traditional plant breeding has been used to provide resistance to pests, disease and other forms of environmental stress, as well as to increase yield and improve upon quality and processing attributes. Over the last decade, the advancement in sequencing technology and bioinformatic analysis has unleashed a wealth of knowledge about chloroplast genetic organization and evolution. The lack of complete plastid genome sequences is one of the major limitations in advancing plastid genetic engineering to other useful crops. This is due to the fact that plastid genome sequences are essential for the identification of endogenous regulatory sequences and optimal …

Studies On The Novel Function Of Amyloid Precursor Protein In Glial Differentiation Of Neural Stem Cells, Young-Don Kwak

Electronic Theses and Dissertations

Although amyloid β (Aβ) deposition has been a hallmark of Alzheimer’s disease (AD), the physiological function of amyloid precursor protein (APP) is not clear. Our results suggested that high concentration of APP induces glial differentiation while physiological level of APP promotes migration and differentiation of neural stem cell (HNSC). HNSCs were mainly differentiated into astrocytes when they are transplanted into APP transgenic mouse brain or treated with a high concentration of secreted-type APP (sAPP) in culture. Staurosporine (STS) induced a distinctive astrocytic morphology in NT-2/D1 neural progenitor cells with expressions of APP and astrocyte-specific markers, glial fibrillary acidic protein (GFAP), …

Phospholipase A2mechanism Of Interfacial Activation,An Interdiscliplinary Approach, Kathleen N. Nemec

Electronic Theses and Dissertations

Phospholipase A2 (PLA2) is an enzyme that hydrolyzes the sn-2-ester bond of membrane phospholipids and liberates arachidonic acid, which is converted to eicosanoids that act as potent mediators of inflammation and allergy. As such this enzyme plays a crucial role in many homeostatic physiological and immunologic processes and disease progression. PLA2s undergo substantial increase in activity upon binding to cellular membranes. This effect of interfacial activation is well recognized, yet its structural and physical aspects are poorly understood. In this work, we have employed the interdisciplinary methods of molecular biology, biochemistry, biophysics, bioinformatics and computational biology, in order to elucidate …

Linking Ligand-Induced Alterations In Androgen Receptor Structure To Differential Gene Expression: A First Step In The Rational Design Of Selective Androgen Receptor Modulators, Dmitri Kazmin, Tatiana Prytkova, C. Edgar Cook, Russell Wolfinger, Tzu-Ming Chu, David Beratan, J. D. Norris, Ching-Yi Chang, Donald P. Mcdonnell

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

We have previously identified a family of novel androgen receptor (AR) ligands that, upon binding, enable AR to adopt structures distinct from that observed in the presence of canonical agonists. In this report, we describe the use of these compounds to establish a relationship between AR structure and biological activity with a view to defining a rational approach with which to identify useful selective AR modulators. To this end, we used combinatorial peptide phage display coupled with molecular dynamic structure analysis to identify the surfaces on AR that are exposed specifically in the presence of selected AR ligands. Subsequently, we …

Heparin Modulates The 99-Loop Of Factor Ixa: Effects On Reactivity With Isolated Kunitz-Type Inhibitor Domains, Pierre F. Neuenschwander, Stephen R. Williamson, Armen Nalian, Kimberly J. Baker-Deadmond

Faculty Publications

Reactivity of factor IXa with basic pancreatic trypsin inhibitor is enhanced by low molecular weight heparin (enoxaparin). Previous studies by us have suggested that this effect involves allosteric modulation of factor IXa. We examined the reactivity of factor IXa with several isolated Kunitz-type inhibitor domains: basic pancreatic trypsin inhibitor, the Kunitz inhibitor domain of protease Nexin-2, and the first two inhibitor domains of tissue factor pathway inhibitor. We find that enhancement of factor IXa reactivity by enoxaparin is greatest for basic pancreatic trypsin inhibitor (>10-fold), followed by the second tissue factor pathway inhibitor domain (1.7-fold) and the Kunitz inhibitor …

Direct Control Of Cell Cycle Gene Expression By Proto-Oncogene Product Actr, And Its Autoregulation Underlies Its Transforming Activity, Maggie C. Louie, Alexey S. Revenko, June X. Zou, Jennifer Yao, Hong-Wu Chen

Collected Faculty Scholarship

ACTR (also called AIB1 and SRC-3) was identified as a coactivator for nuclear receptors and is linked to multiple types of human cancer due to its frequent overexpression. However, the molecular mechanism of ACTR oncogenicity and its function independent of nuclear receptors remain to be defined. We demonstrate here that ACTR is required for both normal and malignant human cells to effectively enter S phase. RNA interference-mediated depletion and chromatin immunoprecipitation assays show that endogenous ACTR directly controls the expression of genes important for initiation of DNA replication, which include cdc6, cdc25A, MCM7, cyclin E, and Cdk2. Moreover, consistent with …