Biochemistry, Biophysics, and Structural Biology Commons^™

Processing The Interspecies Quorum-Sensing Signal Autoinducer-2 (Ai-2) Characterization Of Phospho-(S)-4,5-Dihydroxy-2,3-Pentanedione Isomerization By Lsrg Protein, J. C. Marques, P. Lamosa, Caitlin Elizabeth Russell , '11, R. Ventura, C. D. Maycock, M. F. Semmelhack, Stephen T. Miller, K. B. Xavier

Chemistry & Biochemistry Faculty Works

The molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD) is produced by many different species of bacteria and is the precursor of the signal molecule autoinducer-2 (AI-2). AI-2 mediates interspecies communication and facilitates regulation of bacterial behaviors such as biofilm formation and virulence. A variety of bacterial species have the ability to sequester and process the AI-2 present in their environment, thereby interfering with the cell-cell communication of other bacteria. This process involves the AI-2-regulated lsr operon, comprised of the Lsr transport system that facilitates uptake of the signal, a kinase that phosphorylates the signal to phospho-DPD (P-DPD), and enzymes (like LsrG) that are responsible …

Controlling The Activity Of A Phosphatase And Tensin Homolog (Pten) By Membrane Potential, Jérôme J. Lacroix, Christian R. Halaszovich, Daniela N. Schreiber, Michael G. Leitner, Francisco Bezanilla, Dominik Oliver, Carlos A. Villalba-Galea

School of Pharmacy Faculty Articles

The recently discovered voltage-sensitive phosphatases (VSPs) hydrolyze phosphoinositides upon depolarization of the membrane potential, thus representing a novel principle for the transduction of electrical activity into biochemical signals. Here, we demonstrate the possibility to confer voltage sensitivity to cytosolic enzymes. By fusing the tumor suppressor PTEN to the voltage sensor of the prototypic VSP from Ciona intestinalis, Ci-VSP, we generated chimeric proteins that are voltage-sensitive and display PTEN-like enzymatic activity in a strictly depolarization-dependent manner in vivo. Functional coupling of the exogenous enzymatic activity to the voltage sensor is mediated by a phospholipid-binding motif at the interface between voltage sensor …

The Csc Is Required For Complete Radial Spoke Assembly And Wild-Type Ciliary Motility, Erin E. Dymek, Thomas Heuser, Daniela Nicastro, Elizabeth F. Smith

Dartmouth Scholarship

The ubiquitous calcium binding protein, calmodulin (CaM), plays a major role in regulating the motility of all eukaryotic cilia and flagella. We previously identified a CaM and Spoke associated Complex (CSC) and provided evidence that this complex mediates regulatory signals between the radial spokes and dynein arms. We have now used an artificial microRNA (amiRNA) approach to reduce expression of two CSC subunits in Chlamydomonas. For all amiRNA mutants, the entire CSC is lacking or severely reduced in flagella. Structural studies of mutant axonemes revealed that assembly of radial spoke 2 is defective. Furthermore, analysis of both flagellar beating and …

Characterization Of Esterase Activity From The Bacteria, Francisella Tularensis, The Causative Agent Of Tularemia, Leigh Anna Weston

Undergraduate Honors Thesis Collection

Francisella tularensis is the bacteria responsible for causing the disease tularemia and is listed as one of the top three-biowarfare agents. Among the proteins essential to the virulence and infectivity of F.tularensis are multiple esterases, which are enzymes that break down various ester, thioester, and amide bonds. In this project, the catalytic activity, substrate speci fi city, and structure of a putative esterase from F.tularensis was studied. Latent fluorophores based on the molecule, fluorescein, were unmasked by the enzymatic activity of the esterase and the increase in fluorescence was measured over time to determine how well the e tcrase recognized …

Chiral Recognition Study Of A Bimolecular Process In Amino Acid Chiral Ionic Liquids, Laurel Millikan Heckman

Undergraduate Honors Thesis Collection

In this study, chiral ionic liquid (CIL) solvents were prepared and tested for their chiral discrimination ability by probing with luminescence quenching. The CIL's were also tested for their impurities, viscosity and density. These experiments will help to determine the ability of CIL's to act as solvents in asymmetric catalysis. The chiral ionic liquids in this study were composed of amino acid methyl ester cations and bis(trifluoromethane) sulfonimide or bistri flimide anions (i.e. I-alanine methyl ester bistriflimid e, d-alanine methyl ester bistr iflim ide and I-leuci ne methyl ester bistriflimde). Circularly polarized luminescence (CPL) and its quenching in the chiral …

Digeorge Critical Region 8 (Dgcr8) Is A Double-Cysteine-Ligated Heme Protein., Ian Barr, Aaron T. Smith, Rachel Senturia, Yanqiu Chen, Brooke D. Scheidemantle, Judith N. Burstyn, Feng Guo

Natural Sciences and Mathematics | Faculty Scholarship

All known heme-thiolate proteins ligate the heme iron using one cysteine side chain. We previously found that DiGeorge Critical Region 8 (DGCR8), an essential microRNA processing factor, associates with heme of unknown redox state when overexpressed in Escherichia coli. On the basis of the similarity of the 450-nm Soret absorption peak of the DGCR8-heme complex to that of cytochrome P450 containing ferrous heme with CO bound, we identified cysteine 352 as a probable axial ligand in DGCR8. Here we further characterize the DGCR8-heme interaction using biochemical and spectroscopic methods. The DGCR8-heme complex is highly stable, with a half-life exceeding 4 …

Nogo-B Receptor Is Necessary For Cellular Dolichol Biosynthesis And Protein N-Glycosylation., Kenneth D Harrison, Eon Joo Park, Ningguo Gao, Andrew Kuo, Jeffrey S. Rush, Charles J Waechter, Mark A Lehrman, William C Sessa

Molecular and Cellular Biochemistry Faculty Publications

Dolichol monophosphate (Dol-P) functions as an obligate glycosyl carrier lipid in protein glycosylation reactions. Dol-P is synthesized by the successive condensation of isopentenyl diphosphate (IPP), with farnesyl diphosphate catalysed by a cis-isoprenyltransferase (cis-IPTase) activity. Despite the recognition of cis-IPTase activity 40 years ago and the molecular cloning of the human cDNA encoding the mammalian enzyme, the molecular machinery responsible for regulating this activity remains incompletely understood. Here, we identify Nogo-B receptor (NgBR) as an essential component of the Dol-P biosynthetic machinery. Loss of NgBR results in a robust deficit in cis-IPTase activity and Dol-P production, leading to diminished levels of …

Quorum Sensing In Archaea, Charles Mackin

Honors Scholar Theses

Bacteria coordinate cell density dependent behaviors by communicating through chemical intermediaries in a process called quorum sensing. In a bacterial culture, individual cells will constitutively produce signal molecules, termed autoinducers, and export them into the environment. When the concentration of autoinducers reaches a threshold, the cells sense that they are in a specific situation, which requires the upregulation of certain genes. This upregulation causes the bacteria to produce proteins that allow them to take part in a coordinated population-wide behavior.

In bacteria that are naturally competent, or capable of importing DNA from the environment, the expression of competence genes is …

Design And Bio-Production Of A Nanoparticle Avian Influenza Vaccine, Kashif N. Ather

Honors Scholar Theses

Influenza is one of the most common diseases in the world and the cause for numerous deaths every year. The primary method of combating the disease is the influenza vaccine, which is produced by inoculating chicken eggs with inactivated virus. An emerging solution is to use Self-Assembling Polypeptide Nanoparticles (SAPN) to elicit an immune response in the body, rather than using inactivated viruses. This project focuses on the synthesis, purification, and refolding of two peptide constructs, BN5C and S43, which are specific protein sequences that under the right conditions will refold into the 3-dimensional structures necessary for producing an immune …

Characterizing The Role Of The Bacterial Metallothionein, Smta, In Mammalian Infection, Stephanie R. Davis

Honors Scholar Theses

Mammalian metallothioneins (MT) are induced by various immunomodulatory molecules and are involved in a spectrum of immune processes such as essential metal homeostasis, detoxification of certain heavy metals, inflammation, and immune cell trafficking [1-3]. A bacterial metallothionein, SmtA, shares some sequence homology with mammalian MT as well as its metal-binding capabilities [4]. In addition to its ability to sequester heavy metals, eukaryotic MT has also been shown to scavenge free radicals such as reactive oxygen and nitrogen species (ROS, RNS), interfering with their toxic effects on cells and potentially influencing their regulatory roles in cell proliferation and differentiation [5, 6]. …

Biomarker Signature Classification Of Various Stress Forms, Chana L. Rich

Honors Scholar Theses

Various types of stressful conditions can have unique and important effects on immunity and can lead to dramatic consequences to health. For my University Scholar project, the characteristic biomarker signatures produced from a set of diverse stressors (e.g. psychological, biological and chemical) are being investigated. A biomarker signature is a distinctive biological indicator of a specific condition. High-throughput tools for the measurement of different cellular products have the potential to further our understanding of human disease and facilitated the identification of new biomarkers in all areas of medicine. The hypothesis that each form of stress, psychological, chemical and physical, will …

Characterization Of Arginine-82 Mutants With Non-Native Chromophores, Vivek Alaigh

Honors Scholar Theses

Bacteriorhodopsin, found in most halobacteria, is an integral protein that contains seven transmembrane alpha helices and an organic chromophore, all-trans retinal. Light energy is captured by the protein and results in a series of spectrally discrete intermediates that conclude with a proton being pumped across the membrane from the cytoplasmic side to the extracellular milieu. The most blue-shifted photo-intermediate, the M state, has been of interest for protein-based holographic memory storage devices. Bacteriorhodopsin mutants were prepared with either a 4-hydroxy retinal or 3,4-dihydro retinal analog: R82A, R82C, R82H, R82K, R82N and R82Q. The objective of this research was to investigate …

Expression Of The Porcine Reproductive And Respiratory Syndrome Virus Non-Structural Protein 3 (Nsp 3) In Escherichia Coli, Lidia Beka

Honors Scholar Theses

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is single-stranded, positive-sense RNA virus in the family Arteriviridae, order Nidovirales. PRRSV is the most economically significant viral infection of swine herds in the United States. The single-stranded RNA genome is 15 kb in length and encodes 9 open reading frames (ORF1a, ORF1b, ORF2a, ORF2b and ORFs 3 through 7). ORFs 1a and 1b encode for 13 non-structural proteins (nsp) that are suggested to be involved in transcription and viral genome replication. The exact role of non-structural proteins in PRRSV cycle is still unknown. Moreover, there is a limited availability of reagents such …

Modeling Human Immune Response To The Lyme Disease-Causing Bacteria, Yevhen Rutovytskyy

Honors Scholar Theses

The purpose of this project is to develop and analyze a mathematical model for the pathogen-host interaction that occurs during early Lyme disease.

Based on the known biophysics of motility of Borrelia burgdorferi and a simple model for the immune response, a PDE model was created which tracks the time evolution of the concentrations of bacteria and activated immune cells in the dermis. We assume that a tick bite inoculates a highly localized population of bacteria into the dermis. These bacteria can multiply and migrate. The diffusive nature of the migration is assumed and modeled using the heat equation. Bacteria …

Effects Of Vitamin E Isomer, Gamma Tocotrienol (Gt3), At Inhibiting Cell Growth And Inducing Apoptosis In Colon Cancer Cell Line Hct-116., Havya Dave

Undergraduate Honors Theses

Colorectal cancer is the third most prominent cancer world-wide and it is the second leading cause of cancer deaths in the United States. Many dietary components affect the risk of developing colorectal cancer, such as Vitamin E. Of the eight isomers of Vitamin E, four have a tocotrienol structure. Tocotrienols are found at highest concentrations in palm oil, which is ingested more in areas of Asia where the incidence of colorectal cancer is the lowest, suggesting a role of tocotrienols in the prevention of colorectal cancer. The metabolism of Arachidonic acid pathway produces a host of pro-inflammatory metabolites either by …

Modeling Human Immune Response To The Lyme Disease-Causing Bacteria, Yevhen Rutovytskyy

Honors Scholar Theses

The purpose of this project is to develop and analyze a mathematical model

for the pathogen-host interaction that occurs during early Lyme disease.

Based on the known biophysics of motility of Borrelia burgdorferi and a

simple model for the immune response, a PDE model was created which tracks

the time evolution of the concentrations of bacteria and activated immune

cells in the dermis. We assume that a tick bite inoculates a highly

localized population of bacteria into the dermis. These bacteria can

multiply and migrate. The diffusive nature of the migration is assumed and

modeled using the heat equation. Bacteria …

The Effects Of The Vitamin E Isomers Gamma Tocopherol And Gamma Tocotrienol On The Nfkb Pathway In The Pc-3 Cell Line., Brittney Rudder

Undergraduate Honors Theses

Regions along the Mediterranean and Southern Asia have lower prostate cancer incidence compared to the rest of the world. It has been hypothesized that one of the potential contributing factors for this low incidence includes a higher intake of vitamin E (tocopherols and tocotrienols). This study examines the potential of gamma tocopherol (GT) and gamma tocotrienol (GT3) to reduce prostate cancer proliferation by examining their effects on the NFκB pathway. NFκB is known to inhibit apoptosis in cancer cells. Our data shows that both GT and GT3 are capable of down regulation of NFκB precursors and up regulation of Caspase …

The Effect Of Ultraviolet Light On Cell Viability, Dna Damage And Repair In Hutchinson-Gilford Progeria Syndrome And Bj Fibroblasts., Mckayla Johnson

Undergraduate Honors Theses

Patients of Hutchinson-Gilford Progeria Syndrome (HGPS) display a rate of aging up to ten times that of normal human populations. It might be expected that HGPS cells would have a decreased ability to repair DNA damage through the cell cycle as compared to normal cells such as those of the BJ cell line since DNA damage accumulation is a hallmark phenotype of aging. On earth, we are exposed to far more ultraviolet-B (UV-B, 280-315 nm) and UV-A (315-400 nm) than UV-C (100-280 nm) radiation, since the latter is filtered-out by the atmospheric ozone layer. The relative sensitivity of prematurely aging …

A Predictive Model Which Uses Descriptors Of Rna Secondary Structures Derived From Graph Theory., Alissa Ann Rockney

Electronic Theses and Dissertations

The secondary structures of ribonucleic acid (RNA) have been successfully modeled with graph-theoretic structures. Often, simple graphs are used to represent secondary RNA structures; however, in this research, a multigraph representation of RNA is used, in which vertices represent stems and edges represent the internal motifs. Any type of RNA secondary structure may be represented by a graph in this manner. We define novel graphical invariants to quantify the multigraphs and obtain characteristic descriptors of the secondary structures. These descriptors are used to train an artificial neural network (ANN) to recognize the characteristics of secondary RNA structure. Using the ANN, …

Characterization Of Expression Of The Kcne Gene Family In Zebrafish, Danio Rerio, Lauren Vernlund

Honors Senior Capstones and Theses

The KCNE gene family codes for five transmembrane accessory proteins, "minK related peptides" or Mirps, involved in the modification of voltage-gated potassium (Kv) channels, K+ selective pores vital in the regulation of membrane potential and repolarization in all organisms. In mammals, all five KCNE gene members are conserved and active in the heart. In the zebrafish Danio rerio, there are no apparent orthologs for KCNE2 or KCNE5, yet they contain Kv channels with homologous structure, function, and Mirp regulatory behavior to other organisms. Sequence analysis of wildtype zebrafish KCNE1, 3 and 4, and rtPCR on RNA from zebrafish tissues …