Biochemistry, Biophysics, and Structural Biology Commons^™

Ab Initio Exon Definition Using An Information Theory-Based Approach, Peter K. Rogan

Biochemistry Publications

Transcribed exons in genes are joined together at donor and acceptor splice sites precisely and efficiently to generate mRNAs capa ble of being translated into proteins. The sequence variability in individual splice sites can be modeled using Shannon information theory. In the laboratory, the degree of individual splice site use is inferred from the structures of mRNAs and their relative abundance. These structures can be predicted using a bipartite information theory framework that is guided by current knowledge of biological mechanisms for exon recognition. We present the results of this analysis for the complete dataset of all expressed human exons.

From Gene Mutation To Protein Characterization, David A. Moffet

Chemistry and Biochemistry Faculty Works

A seven-week “gene to protein” laboratory sequence is described for an undergraduate biochemistry laboratory course. Student pairs were given the task of introducing a point mutation of their choosing into the well studied protein, enhanced green fluorescent protein (EGFP). After conducting literature searches, each student group chose the mutation they wanted to introduce into EGFP. Students designed their sequence-specific mutagenic primers and constructed their desired mutation. The resulting EGFP mutant proteins were expressed in E. coli, purified and characterized. This laboratory sequence connected the major concepts of molecular biology and biochemistry, while incorporating the thrill of novel discovery in …

Polymer Microfluidic Devices For Bioanalysis, Xuefei Sun

Theses and Dissertations

Polymeric microchips have received increasing attention in chemical analysis because polymers have attractive properties, such as low cost, ease of fabrication, biocompatibility and high flexibility. However, commercial polymers usually exhibit analyte adsorption on their surfaces, which can interfere with microfluidic transport in, for example, chemical separations such as chromatography or electrophoresis. Usually, surface modification is required to eliminate this problem. To perform stable and durable surface modification, a new polymer, poly(methyl methacrylate-co-glycidyl methacrylate) (PGMAMMA) was prepared for microchip fabrication, which provides epoxy groups on the surface. Whole surface atom transfer radical polymerization (ATRP) and in-channel ATRP approaches were employed to …

Leptospira Interrogans Induces Apoptosis In Macrophages Via Caspase-8- And -3-Dependent Pathways, Dandan Jin, David M. Ojcius, Dexter Sun, Haiyan Dong, Yihui Luo, Yafei Mao, Jie Yan

All Dugoni School of Dentistry Faculty Articles

Apoptosis of host cells plays an important role in modulating the pathogenesis of many infectious diseases. It has been reported that Leptospira interrogans, the causal agent of leptospirosis, induces apoptosis in macrophages and hepatocytes. However, the molecular mechanisms responsible for host cell death remained largely unknown. Here we demonstrate that L. interrogans induced apoptosis in a macrophage-like cell line, J774A.1, and primary murine macrophages in a time- and dose-dependent manner. Apoptosis was associated with the activation of cysteine aspartic acid-specific proteases (caspase-3, caspase-6, and caspase-8), the increased expression of Fas-associated death domain (FADD), and the cleavage of the caspase substrates …

Temperature Influence And Heat Management Requirements Of Microalgae Cultivation In Photobioreactors, Thomas Hagen Mehlitz

Master's Theses

Microalgae are considered one of the most promising feedstocks for biofuel production for the future. The most efficient way to produce vast amounts of algal biomass is the use of closed tubular photobioreactors (PBR). The heat requirement for a given system is a major concern since the best algae growth rates are obtained between 25-30 °C, depending on the specific strain. A procedure to determine temperature influence on algal growth rates was developed for a lab-scale PBR system using the species Chlorella. A maximum growth rate of 1.44 doublings per day at 29 °C (optimal temperature) was determined. In addition, …

Adaptation Of The Bacterial Membrane To Changing Environments Using Aminoacylated Phospholipids, Hervé Roy, Kiley Dare, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Fine‐tuning of the biophysical properties of biological membranes is essential for adaptation of cells to changing environments. For instance, to lower the negative charge of the lipid bilayer, certain bacteria add lysine to phosphatidylglycerol (PG) converting the net negative charge of PG (−1) to a net positive charge in Lys‐PG (+1). Reducing the net negative charge of the bacterial cell wall is a common strategy used by bacteria to resist cationic antimicrobial peptides (CAMPs) secreted by other microbes or produced by the innate immune system of a host organism. The article by Klein et al. in the current issue of …

Structure Of Human Dna Polymerase Κ Inserting Datp Opposite An 8-Oxog Dna Lesion, Rodrigo Vasquez-Del Carpio, Timothy D. Silverstein, Samer Lone, Michael K. Swan, Jayati R. Choudhury, Robert E. Johnson, Satya Prakash, Louise Prakash, Aneel K. Aggarwal

Chemical Sciences Faculty Publications

Background: Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA and 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major lesions formed. It is amongst the most mutagenic lesions in cells because of its dual coding potential, wherein 8-oxoG(syn) can pair with an A in addition to normal base pairing of 8-oxoG(anti) with a C. Human DNA polymerase κ (Polκ) is a member of the newly discovered Y-family of DNA polymerases that possess the ability to replicate through DNA lesions. To understand the basis of Polκ's preference for insertion of an A opposite 8-oxoG lesion, …

A Watershed Analysis Of Salmon Lake And Mcgrath Pond: Implications For Water Quality And Land Use Management, Colby Environmental Assessment Team, Colby College, Problems In Environmental Science Course (Biology 493), Colby College

Colby College Watershed Study: Salmon Lake and McGrath Pond (2009, 1993)

In the summer and fall of 2009, the Colby Environmental Assessment Team (CEAT) studied the water quality of Salmon Lake and McGrath Pond, located in the Belgrade Lakes Region of Maine. The physical, chemical and biological characteristics of water quality were measured and analyzed to evaluate the current health of these lakes. Water quality data collected during the summer and fall of 2009 were compared with data from previous years to study the historic water quality trends. Land use patterns in the Salmon/McGrath watershed were also examined to investigate their impact on the lake water quality. The water quality trends …

Nerve, Muscle, Blood, Toil, Tears, And Sweat: England’S Pioneering Biophysicist, Soldier, And Statesman, Arshad Khan

Selected Works Temporary Series

No abstract provided.

Liposomes Recruit Ipac To The Shigella Flexneri Type Iii Secretion Apparatus Needle As A Final Step In Secretion Induction, C. R. Epler, Nicholas E. Dickenson, A. J. Olive, W. L. Picking, W. D. Picking

Chemistry and Biochemistry Faculty Publications

Shigella flexneri contact with enterocytes induces a burst of protein secretion via its type III secretion apparatus (TTSA) as an initial step in cellular invasion. We have previously reported that IpaD is positioned at the TTSA needle tip (M. Espina et al., Infect. Immuno. 74:4391-4400, 2006). From this position, IpaD senses small molecules in the environment to control the presentation of IpaB to the needle tip. This step occurs without type III secretion induction or IpaC recruitment to the S. flexneri surface. IpaC is then transported to the S. flexneri surface when target cell lipids are added, and this event …

Influence On Proline Upon The Folding And Geometry Of A Model Transmembrane Peptide, Rachel Thomas

Inquiry: The University of Arkansas Undergraduate Research Journal

The orientations, geometries and lipid interactions of designed transmembrane (TM) peptides have attracted significant experimental and theoretical interest. Because the amino acid proline will introduce a known discontinuity into an alpha-helix, it is important to measure the extent of helix kinking caused by a single proline within an isolated TM helical domain. For this purpose, acetyl-GWWLALALAP10ALALALWWA-ethanolamide was synthesized, and pairs of deuterated alanines were included by using 60-100% deuterated fluorenylmethoxycarbonyl-L-alanine ifmoc-d4-L-Ala) at selected sequence positions. Solid-state deuterium fH) magnetic resonance spectra from oriented, hydrated samples (1140, peptide/lipid; using several lipids) reveal signals from many of the alanine backbone Ca deuterons …

Loop Dynamics Of The Extracellular Domain Of Human Tissue Factor And Activation Of Factor Viia, Agnese S. Minazzo, Reuben C. Darlington, J. B. A. Ross

Chemistry and Biochemistry Faculty Publications

In the crystal structure of the complex between the soluble extracellular domain of tissue factor (sTF) and activesite- inhibited VIIa, residues 91 and 92 in the Pro⁷⁹-Pro⁹² loop of sTF interact with the catalytic domain of VIIa. It is not known, however, whether this loop has a role in allosteric activation of VIIa. Time-resolved fluorescence anisotropy measurements of probes covalently bound to sTF mutants E84C and T121C show that binding uninhibited Factor VIIa affects segmental motions in sTF. Glu⁸⁴ resides in the Pro⁷⁹-Pro⁹² loop, and Thr¹²¹ resides in the turn between the …

Emissions From Biomass Burning In The Yucatan, Robert J. Yokelson, J. D. Crounse, P. F. Decarlo, T. Karl, S. P. Urbanski, E. Atlas, T. Campos, Y. Shinozuka, V. Kasputin, A. D. Clarke, A. Weinheimer, D. J. Knapp, D. D. Montzka, J. Holloway, P. Weibring, F. Flocke, W. Zheng, D. Toohey, P. O. Wennberg, C. Wiedinmyer, L. Mauldin, A. Fried, D. Richter, J. Walega, J. L. Jimenez, K. Adachi, P. R. Buseck, S. R. Hall, R. Shetter

Chemistry and Biochemistry Faculty Publications

In March 2006 two instrumented aircraft made the first detailed field measurements of biomass burning (BB) emissions in the Northern Hemisphere tropics as part of the MILAGRO project. The aircraft were the National Center for Atmospheric Research C-130 and a University of Montana/US Forest Service Twin Otter. The initial emissions of up to 49 trace gas or particle species were measured from 20 deforestation and crop residue fires on the Yucatan peninsula. This included two trace gases useful as indicators of BB (HCN and acetonitrile) and several rarely, or never before, measured species: OH, peroxyacetic acid, propanoic acid, hydrogen peroxide, …

Biomass Burning And Urban Air Pollution Over The Central Mexican Plateau, J. D. Crounse, P. F. Decarlo, Donald R. Blake, L. K. Emmons, T. L. Campos, E. C. Apel, A. D. Clarke, A. J. Weinheimer, D. C. Mccabe, Robert J. Yokelson, J. L. Jimenez, P. O. Wennberg

Chemistry and Biochemistry Faculty Publications

Observations during the 2006 dry season of highly elevated concentrations of cyanides in the atmosphere above Mexico City (MC) and the surrounding plains demonstrate that biomass burning (BB) significantly impacted air quality in the region. We find that during the period of our measurements, fires contribute more than half of the organic aerosol mass and submicron aerosol scattering, and one third of the enhancement in benzene, reactive nitrogen, and carbon monoxide in the outflow from the plateau. The combination of biomass burning and anthropogenic emissions will affect ozone chemistry in the MC outflow.

Nerve, Muscle, Blood, Toil, Tears, And Sweat: England’S Pioneering Biophysicist, Soldier, And Statesman, Khan

Departmental Papers (Biology)

No abstract provided.

Mass Stranding Of Marine Birds Caused By A Surfactant-Producing Red Tide., David A. Jessup, Melissa A. Miller, John P. Ryan, Hannah M. Nevins, Heather A. Kerkering, Abdou Mekebri, David B. Crane, Tyler A. Johnson, Raphael M. Kudela

Natural Sciences and Mathematics | Faculty Scholarship

In November-December 2007 a widespread seabird mortality event occurred in Monterey Bay, California, USA, coincident with a massive red tide caused by the dinoflagellate Akashiwo sanguinea. Affected birds had a slimy yellow-green material on their feathers, which were saturated with water, and they were severely hypothermic. We determined that foam containing surfactant-like proteins, derived from organic matter of the red tide, coated their feathers and neutralized natural water repellency and insulation. No evidence of exposure to petroleum or other oils or biotoxins were found. This is the first documented case of its kind, but previous similar events may have gone …

Proteomic Analysis Of Endodontic Infections By Liquid Chromatography–Tandem Mass Spectrometry, R. Nandakumar, Nandakumar Madayiputhiya, A. F. Fouad

Department of Biochemistry: Faculty Publications

Introduction—Endodontic infections are very prevalent and have a polymicrobial etiology characterized by complex interrelationships between endodontic microorganisms and the host defenses. Proteomic analysis of endodontic infections can provide global insights into the invasion, pathogenicity mechanisms, and multifactorial interactions existing between root canal bacteria and the host in the initiation and progression of apical periodontitis. The purpose of this study was to apply proteomic techniques such as liquid chromatography–tandem mass spectrometry (LC–MS/ MS) for the identification of proteins of bacterial origin present in endodontic infections.

Methods—Endodontic specimens were aseptically obtained from seven patients with root canal infections. Protein mixtures were subjected …

Hyaluronidase Activity Of Human Hyal1 Requires Active Site Acidic And Tyrosine Residues, Ling Zhang, Alamelu G. Bharadwaj, Andrew Casper, Joel Barkley, Joseph J. Barycki, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

Hyaluronidases are a family of endolytic glycoside hydrolases

that cleave the β1–4 linkage between N-acetylglucosamine and

glucuronic acid in hyaluronan polymers via a substrate-assisted

mechanism. In humans, turnover of hyaluronan by this enzyme

family is critical for normal extracellular matrix remodeling.

However, elevated expression of the Hyal1 isozyme accelerates

tumor growth and metastatic progression. In this study, we used

structural information, site-directed mutagenesis, and steady

state enzyme kinetics to probe molecular determinants of

human Hyal1 function. Mutagenesis of active site residues

Glu¹³¹ and Tyr²⁴⁷ to Gln and Phe, respectively, eliminated activity

at all hyaluronan concentrations (to 125 …

Mechanistic Details Of Glutathione Biosynthesis Revealed By Crystal Structures Of Saccharomyces Cerevisiae Glutamate Cysteine Ligase, Ekaterina I. Biterova, Joseph J. Barycki

Department of Biochemistry: Faculty Publications

Glutathione is a thiol-disulfide exchange peptide critical for

buffering oxidative or chemical stress, and an essential cofactor

in several biosynthesis and detoxification pathways. The ratelimiting

step in its de novo biosynthesis is catalyzed by glutamate

cysteine ligase, a broadly expressed enzyme for which limited

structural information is available in higher eukaryotic species.

Structural data are critical to the understanding of clinical glutathione

deficiency, as well as rational design of enzyme modulators

that could impact human disease progression. Here, we

have determined the structures of Saccharomyces cerevisiae glutamate

cysteine ligase (ScGCL) in the presence of glutamate and

MgCl2 (2.1 Å; R …

Modulation Of The Heme Electronic Structure And Cystathionine Β-Synthase Activity By Second Coordination Sphere Ligands: The Role Of Heme Ligand Switching In Redox Regulation, Sangita Singh, Peter Madzelan, Jay Stasser, Colin L. Weeks, Donald F. Becker, Thomas G. Spiro, James Penner-Hahn, Ruma Banerjee

Department of Biochemistry: Faculty Publications

In humans, cystathionine β-synthase (CBS) is a hemeprotein, which catalyzes a pyridoxal phosphate (PLP)-dependent condensation reaction. Changes in the heme environment are communicated to the active site, which is ~20 Å away. In this study, we have examined the role of H67 and R266, which are in the second coordination sphere of the heme ligands, H65 and C52 respectively, in modulating the heme's electronic properties and in transmitting information between the heme and active sites. While the H67A mutation is comparable to wild-type CBS, interesting differences are revealed by mutations at the R266 site. The pathogenic mutant, R266K, is moderately …

Heme Regulation Of Human Cystathionine Β-Synthase Activity: Insights From Fluorescence And Raman Spectroscopy, Colin L. Weeks, Sangita Singh, Peter Madzelan, Ruma Banerjee, Thomas G. Spiro

Department of Biochemistry: Faculty Publications

Cystathionine β-synthase (CBS) plays a central role in cysteine metabolism, and malfunction of the enzyme leads to homocystinuria, a devastating metabolic disease. CBS contains a pyridoxal 5′- phosphate (PLP) cofactor which catalyzes the synthesis of cystathionine from homocysteine and serine. Mammalian forms of the enzyme also contain a heme group, which is not involved in catalysis. It may, however, play a regulatory role, since the enzyme is inhibited when CO or NO are bound to the heme. We have investigated the mechanism of this inhibition using fluorescence and resonance Raman spectroscopies. CO binding is found to induce a tautomeric shift …

Molecular Dynamics Study Of Single Stranded Peptide Nucleic Acids, Anna K. Manukyan

Theses and Dissertations in Biomedical Sciences

A PNA molecule is a DNA strand where the sugar-phosphate backbone has been replaced by a structurally homomorphous pseudopeptide chain consisting of N(2-aminoethyl)-glycine units. PNA binds strongly to both DNA and RNA. However, an analysis of the X-ray and NMR data show that the dihedral angles of PNA/DNA or PNA/DNA complexes are very different from those of DNA:DNA or RNA:RNA complexes. In addition, the PNA strand is very flexible. One way to improve the binding affinity of PNA for DNA/RNA is to design a more pre-organized PNA structure. An effective way to rigidify the PNA strand is to introduce …

Influence Of Irradiance And Iron On The Growth Of Colonial Phaeocystic Antarctica: Implications For Seasonal Bloom Dynamics In The Ross Sea, Antarctica, Nathan S. Garcia, Peter N. Sedwick, Giacomo R. Ditullio

OES Faculty Publications

Laboratory culture experiments were used to investigate the growth rate of colonial Phaeocystis anarctica as a function of irradiance and dissolved iron concentration. The experiments were conducted with a P. antarctica strain isolated from the southern Ross Sea, Antarctica, and made use of natural, low-iron (P. antarctica attained an average maximum cell-specific growth rate of 0.37 d^-1at an irradiance of 68 μE m^-2s^-1, above which growth rates decreased to 0.27 d^-1 at an irradiance of 314 μE m^-2s^-1. The dependence of growth rate on ambient dissolved iron concentration was …

Isothermal Titration Calorimetry Studies Of Protein-Mediated Interactions And Preliminary Structural Studies Of Tandem Pdz1-2 Domain Of Psd-95 Protein, Ana Jankovic

Wayne State University Dissertations

Protein-mediated interactions that involve multiple ligands in their binding mechanisms are critical for many cellular functions. The primary focus of this dissertation research was to investigate such interactions for two proteins, the PDZ domain and frataxin, involving peptide and metal binding ligands, respectively. The three component projects of this work comprised (1) thermodynamic analysis of PDZ domain binding using calorimetry; (2) X-ray crystallographic structural studies of a PDZ dual domain; and (3) thermodynamic analysis of frataxin binding to iron. The specific goal of the research conducted with the PDZ domains was to understand the mechanism of action of multiple/tandem protein …

Role Of Cyp2a5 In Drug Metabolism, Chemical Toxicity, And Maintenance Of Steroid Hormone Homeostasis : Insights From Studies On A Novel Cyp2a5-Null Mouse Model, Xin Zhou

Legacy Theses & Dissertations (2009 - 2024)

The central hypothesis is that CYP2A5 plays an important role in the metabolism of xenobiotic substrates, and in the toxicity induced by over-exposure to drugs, as well as in the metabolism of endogenous compounds and regulation of steroid hormone homeostasis. The specific aims are: 1) to generate and characterize a Cyp2a5-null mouse; 2) to determine the role of CYP2A5 in the systemic clearance of nicotine and cotinine; and 3) to explore the mechanisms underlying the resistance of the lateral nasal gland (LNG) of male Cyp2g1-null/Cyp2a5-low mouse and Cyp2a5-null mouse to acetaminophen (AP) toxicity.

Genetically Engineered Repetitive Polypeptides : Design, Synthesis, Characterization, And Applications, Natalya I. Topilina

Legacy Theses & Dissertations (2009 - 2024)

This work is dedicated to the investigation of protein folding with the emphasis on the aggregation and amyloid fibrils formation. The focus of the research is to elucidate the relationship between polypeptide chain sequence and folding/aggregation properties. For this purpose, i) libraries of the de novo macromolecular repetitive β-sheet forming polypeptides were designed, and ii) the strategy for their rapid creation was developed. The strategy enables retention of a basic polypeptide core, yet allowing variations in the repetitive sequence, degree of polymerization and distribution of specific amino acids. The polypeptides were expressed in E. coli, purified, and characterized by various …

Secondary Structures And Thermodynamic Properties Of Ampa Receptor Aptamers, Sabarinath Jayaseelan

Legacy Theses & Dissertations (2009 - 2024)

Using systematic evolution of ligands by exponential enrichment (SELEX), our lab previously selected a class of competitive RNA aptamers against the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. One aptamer in this class was found to have a minimal, functional sequence of 58 nucleotides. In vitro transcription generates two RNA transcripts with the same sequence, which we named M1 and M2, both of which are required to work together to inhibit the AMPA receptors. Although the M1 and M2 species have the same sequence, they cannot be interconverted through unfolding by denaturation/refolding by renaturation. To probe the secondary structures and the thermodynamic …

The Recognition And Folding Of Ncp7 (Nucleocapsid Protein) With Tardna And Psi Site Rna Hairpin Structures From Hiv-1 : A Spin Label Study, Yan Sun

Legacy Theses & Dissertations (2009 - 2024)

The nucleocapsid (NC) protein NCp7 of the human immunodeficiency virus type 1(HIV 1) is a small basic protein with two zinc finger motifs. NCp7 has key roles in virus replication and structure, which rely on its interactions with nucleic acids. We investigated the interaction of NCp7 with Transactivation Response Element (TAR) DNA which is critical for minus strand transfer during the HIV 1 reverse transcription. The EPR spin probe nitroxide was used as the reporter. Binding of NCp7 to TARDNA caused TARDNA condensation, as inferred from the probe tumbling time which markedly increased to several nanoseconds at a 4:1 NCp7 …

Regulation Of Intracellular Calcium Concentration By Nanosecond Pulsed Electric Fields, Shaka S. Scarlett, Jody A. White, Peter F. Blackmore, Karl H. Schoenbach, Juergen Kolb

Bioelectrics Publications

Changes in [Ca²⁺]_i response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca²⁺]_i was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca²⁺]_i response was due to the release of Ca²⁺ from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca²⁺]_i was due to both internal release and to influx across the plasma …

Asymmetric Transfer Hydrogenation Of Allylic Alcohols With Homogeneous Chiral Ruthenium Catalysts: A Proposed Mechanism, Marie G. Beauchamps

Seton Hall University Dissertations and Theses (ETDs)

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