Biochemistry, Biophysics, and Structural Biology Commons^™

Boreal Forest Fire Emissions In Fresh Canadian Smoke Plumes: C-1-C-10 Volatile Organic Compounds (Vocs), Co2, Co, No2, No, Hcn And Ch3cn, Isobel J. Simpson, S. K. Akagi, B. Barletta, N. J. Blake, Y. Choi, G. S. Diskin, A. Fried, H. E. Fuelberg, S. Meinardi, F. S. Rowland, S. A. Vay, A. J. Weinheimer, P. O. Wennberg, P. Wiebring, A. Wisthaler, M. Yang, Robert J. Yokelson, Donald R. Blake

Chemistry and Biochemistry Faculty Publications

Boreal regions comprise about 17% of the global land area, and they both affect and are influenced by climate change. To better understand boreal forest fire emissions and plume evolution, 947 whole air samples were collected aboard the NASA DC-8 research aircraft in summer 2008 as part of the ARCTAS-B field mission, and analyzed for 79 non-methane volatile organic compounds (NMVOCs) using gas chromatography. Together with simultaneous measurements of CO2, CO, CH4, CH2O, NO2, NO, HCN and CH3CN, these measurements represent the most comprehensive assessment of trace gas emissions from boreal forest fires to date. Based on 105 air samples …

Nmr Solution Structures Of Two Hairpins Of E. Coli 16s Rrna: The Effects Of Mutations And Chemical Modifications On Structure And Function Of Rrna, Yu Liu

Wayne State University Dissertations

The structures of two functional mutants, the UC (G690U, U697C) and the QM mutants (G690A, G693C, A695C, U697A) of the 690 hairpin of E. coli 16S ribosomal RNA were determined by NMR. The UC mutant and the QM mutant with high biological function are able to fold into structures that are isomorphous with the wild-type 690 hairpin sequence. The structural comparisons among the functional mutants and the wild-type provides structural validation for previously identified specific functional groups that are crucial for maintaining function of the 690 hairpin. The key groups for maintaining the structure and function of the 690 loop …

Exploring Potential Drug Target Sites In The Ribosome Using Cisplatin And Its Analogues, Keshab Rijal

Wayne State University Dissertations

Cis-diamminodichloridoplatinum (II), cisplatin, is an antitumor drug that has been used to treat several types of cancers. The reaction of cisplatin with DNA has been studied and discussed extensively in the literature; however, the effects of cisplatin on RNA function are poorly understood. In this thesis, two aspects of cisplatin, its preferred sites of interaction with RNA and its use as a chemical probe to gain accessibility information, were explored.

To understand the site-selectivity of cisplatin with RNA, model RNA constructs and full-length 16S rRNA were employed. The binding studies revealed a cisplatin preference for guanosine-rich sequences. Primer extensions in …

Study Of Protein-Rna Interactions Using Fluorescence Resonance Energy Transfer (Fret) And Single-Molecule Fret, Rajan Lamichhane

Wayne State University Dissertations

In the cell, RNA and protein, interact to form ribonucleoprotein complexes (RNPs) that have vital structural, catalytic and regulatory roles. Despite their functional importance, the mechanistic details and dynamics of RNPs are poorly understood. Single-molecule Fluorescence Resonance Energy Transfer (smFRET) techniques that provide information about heterogeneity and dynamic behaviors of molecules have been developed to investigate inter- and intra-molecular interactions. Here we have used FRET in combination with smFRET to study three very different RNP systems.

Alternative splicing is a highly regulated biological process that plays a crucial role in proteomic diversity in eukaryotes. One splicing regulator, PTB, has been …

The Role Of The Sparc Acidic Domain And Egf-Like Module In Glioma Migration, Invasion, And Signaling, Heather M. Mcclung

Wayne State University Dissertations

THE ROLE OF THE SPARC ACIDIC DOMAIN AND EGF-LIKE MODULE IN GLIOMA MIGRATION, INVASION, AND SIGNALING

HEATHER M. MCCLUNG

Advisor: Sandra A. Rempel, Ph.D.

Major: Pharmacology

Degree: Doctor of Philosophy

We have previously shown that Secreted Protein Acidic and Rich in Cysteine (SPARC) is upregulated in all astrocytoma grades and increases tumor cell migration and invasion. It is thought that different domains within the protein may regulate SPARC functions, suggesting domain-specific targeting to inhibit invasion. To enhance our understanding of SPARC-mediated invasion, we first confirm, at the protein level, our previous cDNA array results, that SPARC increases expression of the …

Characterization Of Splicing Mechanisms By Single-Molecule Fluorescence, Krishanthi Sanjeewani Karunatilaka

Wayne State University Dissertations

Group II introns rank amongst the largest self-splicing ribozymes found in bacteria and organellar genomes of various eukaryotes. Despite the diversity in primary sequences, group II introns posses highly conserved secondary structures consisting of six domains (D1-D6). To perform its function, the large multidomain group II intron RNA must adopt the correctly folded structure. As a result, in vitro splicing of these introns requires high ionic strength and elevated temperatures. In vivo, this process is mainly assisted by protein cofactors. However, the exact mechanism of protein-mediated splicing of group II intron RNA is still not known.

In order to …

Palmitoylation And The Yeast Casein Kinase Yck2, Irene Papanayotou

Wayne State University Dissertations

Palmitoylation is a post-translational lipid modification that allows proteins to interact with membranes. In the yeast Saccharomyces cerevisiae, the casein kinase Yck2 is palmitoylated twice at its two C-terminal palmitoyl-accepting cysteine residues, by the palmitoyl-transferring enzyme Akr1. Once palmitoylated, Yck2 traffics through the well characterized secretory pathway to the plasma membrane where it participates in many cellular functions, including bud morphogenesis, cytokinesis, nutrient sensing, and receptor internalization. While the hydrophilic Yck2 is presumably synthesized on cytosolic ribosomes, it gains access to the membrane system by interaction with the six transmembrane-spanning Golgi-localized Akr1. Since palmitoylation occurs at membranes and the palmitoyl-transferases …

Identification Of The Role Of The Sal Locus In Streptococcus Pyogenes Virulence During Host-Pathogen Interactions, Phanramphoei Namprachan-Frantz

Wayne State University Dissertations

The pathogenesis of Streptococcus pyogenes is due to its ability to overcome and adapt to the harsh environment created by the host immune response. The focus of this project was the SalKR two-component regulatory system, which facilitates bacterial adaptation by responding to environmental signals during host pathogen-interactions. The first goal of this project was to determine a role in virulence for the SalKR regulatory system. The complete deletion of the salKR genes in the wild type S. pyogenes strain HSC5 produced a highly attenuated mutant in a Zebrafish infection model. The ΔsalKR mutant appeared to lose the ability to survive …

Laser-Assisted Single-Molecule Refolding, Rui Zhao

Wayne State University Dissertations

Non-coding RNAs must fold into precise secondary and tertiary structures in order to perform the biological functions. Due to the flexibility of RNA, the RNA folding energy landscape can be rugged and full of local minimum (kinetic trap). To provide a means to study kinetically trapped RNAs, we have developed a new technique combining single-molecule FRET detection with laser induced temperature jump. We have calibrated the magnitude of the temperature jump with 1˚C accuracy using gold micro-size sensor. The accuracy of temperature calibration was confirmed by close agreement between single-molecule and bulk DNA duplex melting experiments.

HIV 1 DIS RNAs …

Artificial And Natural Nucleic Acid Self Assembling Systems, Marcus Wood

Wayne State University Dissertations

Nucleic acids are good candidates for nanomachine construction. They participate in all the processes of life, and so can function as structural building blocks and dynamic catalysts. However, to use nucleic acids as nanomachines, a better understanding of their material properties, how to design structures using them, and their dynamics is needed. We have tried to address these issues, in a small way, with nucleic acid force field development, an attempt at nanostructural design and synthesis using DNA, and a study of the RNA/protein regulatory dynamics of the tryptophan regulatory attenuation protein.

Development Of Chemical Inducers Of Dimerization For Screening Competitive Histone Deactelyase Inhibitors, Emily Lynn Aubie

Wayne State University Dissertations

Histone Deacetylase (HDAC) proteins are transcriptional regulators that affect histone proteins, which are involved in packaging of DNA into chromosomes. HDACs have been linked to the proliferation of cancer through their role in transcriptional regulation. Due to these findings, HDAC inhibitors have been explored as anti-cancer agents. Several HDAC inhibitors are currently in various stages of clinical trials, and the inhibitor suberoyl anilide hydroxamic acid (SAHA) has been FDA approved for treatment of cutaneous T-Cell lymphoma. Currently, most of the known HDAC inhibitors are non-selective, which causes non-specific binding to the active sites of all HDAC isoforms, including those not …

Hedgehog Signaling: A Potential Therapeutic Target For Non-Small Cell Lung Cancer, Ma'in Yehya Maitah

Wayne State University Dissertations

The American Cancer Society estimated that 222,520 Americans were diagnosed with lung cancer and 157,300 died of lung cancer in 2010 (Jemal et al. 2009, 225-249;Jemal et al. 2011, 69-90). The clinical outcome of patients diagnosed with non-small cell lung cancer (NSCLC), the major lung cancer sub-types, is very poor, which calls for innovative research for finding novel therapeutic targets and agents for better treatment outcome.

Emerging evidences have suggested that a phenomenon called Epithelial-to-Mesenchymal Transition (EMT), which shares similar molecular characteristics with cancer stem-like cells, contributes to lung cancer treatment failure. In view of the fact that EMT process …

Mne1 Is A Novel Component Of The Mitochondrial Splicing Apparatus Responsible For Processing Of A Cox1 Group I Intron In Yeast, Talina Watts, Oleh Khalimonchuk, Rachel Z. Wolf, Edward M. Turk, Georg Mohr, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Saccharomyces cerevisiae cells lacking Mne1 are deficient in

intron splicing in the gene encoding the Cox1 subunit of cytochrome

oxidase but contain wild-type levels of the bc1 complex.

Thus, Mne1 has no role in splicing of COB introns or expression

of the COB gene. Northern experiments suggest that splicing of

the COX1 aI5β intron is dependent on Mne1 in addition to the

previously known Mrs1, Mss116, Pet54, and Suv3 factors. Processing

of the aI5_ intron is similarly impaired in mne1∆ and

mrs1∆ cells and overexpression of Mrs1 partially restores the

respiratory function of mne1∆ cells. Mrs1 …

Development Of Biomarkers For Endocrine Disrupting Compounds In The Fathead Minnow, Pimephales Promelas, Tara Frare

Undergraduate Review

Organic waste compounds (OWC’s) are an environmental concern that can have adverse effects on humans, animals, and the environment. Ethinyl estradiol (EE2) is one common OWC and is an active ingredient in oral birth control pills. The presence of EE2 in aquatic environments and drinking water can be linked to decreased fertility and altered physiology in fish. I am developing Q-PCR biomarkers to detect changes in fish at low concentrations before adverse effects occur at higher concentrations. RNA was extracted from twenty fathead minnows that had not been exposed to OWC’s and will be used to synthesize cDNA. Thus far …

Method Development And Applications To Screening And Characterization Of Rrna-Targeting Small Molecules, Papa Nii Asare-Okai

Wayne State University Dissertations

A series of single ring aminoglycoside analogues was tested for binding to a model RNA representing the A site using electrospray ionization mass spectrometry (ESI-MS). Several of the synthetic analogues with low molecular weights were found to bind to the RNA with affinities comparable to the parental aminoglycoside neamine, with apparent dissociation constants in the low micromolar range. Salt dependence of the affinity constants for the single ring analogues revealed a predominantly electrostatic binding mode. Footprinting experiments revealed that one of the compounds (DHR23) has a similar binding site as the antibiotic paromomycin. DMS chemical probing results also suggest that …

Mechanisms Of Alpha]-Synuclein-Induced Neurodegenertaion In Parkinson's Disease And Stroke, Cherine Belal

Electronic Theses and Dissertations

Parkinson’s disease (PD) is a debilitating neurodegenerative disorder affecting one million Americans. Despite its social and economic impact, the pathological cascades that lead to neuron dysfunction and degeneration in PD are poorly understood. Endoplasmic reticulum (ER) stress has been implicated as an initiator or contributing factor in neurodegenerative diseases including PD. The ER is an organelle central to protein folding and intracellular Ca2+ homeostasis. Perturbations of these functions result in ER stress and upregulation of ER stress proteins, of which some have been implicated in counteracting ER stress-induced cell death. The mechanisms that lead to ER stress and how ER …

Molecular Details Of The Mitochondrial Iron Sulfur Cluster Assembly Pathway, Swati Rawat

Wayne State University Dissertations

MOLECULAR DETAILS OF THE MITOCHONDRIAL IRON SULFUR CLUSTER ASSEMBLY PATHWAY

Iron-sulfur clusters are an important class of prosthetic group involved in electron transfer, enzyme catalysis, and regulation of gene expression. Their biosynthesis requires complex machinery located within the mitochondrion since free iron and sulfide are extremely toxic to the cell. Defects in this pathway results in several diseases such as Friedreich's Ataxia (FRDA), Sideroblastic Anemia and ISCU Myopathy. Therefore molecular details of the biogenesis pathway will provide deep insight in the pathway and treatment options for these diseases. FRDA is caused by deficiency of a single protein called as `Frataxin'. …

The Incidence Of Type-1 Diabetes In Nod Mice Is Modulated By Restricted Flora Not Germ-Free Conditions., Cecile King, Nora Sarvetnick

Journal Articles: Regenerative Medicine

In the NOD mouse, the incidence of type-1 diabetes is thought to be influenced by the degree of cleanliness of the mouse colony. Studies collectively demonstrate that exposure to bacterial antigen or infection in the neonatal period prevents diabetes [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], supporting the notion that immunostimulation can benefit the maturation of the postnatal immune system [11]. A widely accepted extrapolation from this data has been the notion that NOD mice maintained under germ-free conditions have an increased incidence of diabetes. However, evidence supporting this influential concept is surprisingly limited [12]. In this …

Cam Kinase Regulation Of Akt And Bad In Prostate Cancer Cells, John M. Schmitt, Samantha F. Smith

Faculty Publications - Department of Biological & Molecular Science

Abstract AKT and its substrate BAD promote prostate cancer cell survival. Agonists, such as carbachol, and hormones that increase intracellular calcium concentration can activate AKT leading to cancer cell survival. LNCaP prostate cancer cells express the carbachol-sensitive M3-subtype of GPCR’s that increase intracellular calcium and activate the family of Ca2+/Calmodulin-dependent Protein Kinases (CaM Ks). One type of CaM Kinase, CaM Kinase Kinase (CaM KK), directly phosphorylates AKT on threonine 308. AKT phosphorylation and activation can enhance cell survival through phosphorylation BAD protein and the subsequent blockade of caspase activation. Our goals were to examine the mechanism of carbachol activation of …

Trp Channel Regulation Of Estrogen Signaling, John M. Schmitt, Jessica N. Magill

Faculty Publications - Department of Biological & Molecular Science

Abstract Calcium regulates numerous cell functions including growth and development. Calcium can enter cells through transient receptor potential channels (TRPCs). Previous studies in MCF-7 cells have suggested that the expression of one particular TRPC, TRPC6, correlates with cell transformation and disease progression. Calcium has several cellular targets including the Calcium/Calmodulin-dependent protein kinases (CaM Ks) and ERK. Previous work has shown that estrogen (E2) may utilize CaM Ks and ERK to promote breast cancer cell proliferation, however the possible involvement of TRPCs in this pathway is currently unknown. Our objective was to understand which E2 receptor is used in our system …

Disorder Predictors Also Predict Backbone Dynamics For A Family Of Disordered Proteins, Gary W. Daughdrill, Wade M. Borcherds, Hongwei Wu

Molecular Biosciences Faculty Publications

Several algorithms have been developed that use amino acid sequences to predict whether or not a protein or a region of a protein is disordered. These algorithms make accurate predictions for disordered regions that are 30 amino acids or longer, but it is unclear whether the predictions can be directly related to the backbone dynamics of individual amino acid residues. The nuclear Overhauser effect between the amide nitrogen and hydrogen (NHNOE) provides an unambiguous measure of backbone dynamics at single residue resolution and is an excellent tool for characterizing the dynamic behavior of disordered proteins. In this report, we show …

Crystallization And Preliminary X-Ray Crystallographic Analysis Of Ligand-Free And Arginine-Bound Forms Of Thermotoga Maritima Arginine-Binding Protein, Alessia Ruggiero, Jonathan D. Dattelbaum, Anna Pennacchio, Luisa Iozzion, Maria Staiano, Matthew S. Luchansky, Bryan S. Der, Rita Berisio, Sabato D'Auria, Luigi Vitagliano

Chemistry Faculty Publications

The arginine-binding protein from Thermotoga maritima (TmArgBP) is an arginine-binding component of the ATP-binding cassette (ABC) transport system in this hyperthermophilic bacterium. This protein is endowed with an extraordinary stability towards thermal and chemical denaturation. Its structural characterization may provide useful insights for the clarification of structure– stability relationships and for the design of new biosensors. Crystallization trials were set up for both arginine-bound and ligand-free forms of TmArgBP and crystals suitable for crystallographic investigations were obtained for both forms. Ordered crystals of the arginine adduct of TmArgBP could only be obtained by using the detergent LDAO as an additive …

Dynamic Light Scattering And Zeta Potential Of Colloidal Mixtures Of Amelogenin And Hydroxyapatite In Calcium And Phosphate Rich Ionic Milieus, Vuk Uskoković, Roselyn Odsinada, Sonia Djordjevic, Stefan Habelitz

Pharmacy Faculty Articles and Research

The concept of zeta-potential has been used for more than a century as a basic parameter in controlling the stability of colloidal suspensions, irrespective of the nature of their particulate ingredients – organic or inorganic. There are prospects that self-assembly of peptide species and the protein-mineral interactions related to biomineralization may be controlled using this fundamental physicochemical parameter. In this study, we have analyzed the particle size and zeta-potential of the full-length recombinant human amelogenin (rH174), the main protein of the developing enamel matrix, in the presence of calcium and phosphate ions and hydroxyapatite (HAP) particles. As calcium and phosphate …

Quantitative Proteomics Analysis Of Differentially Expressed Proteins In Aβ(17-42) Treated Synaptosomes, Jaffer Mohammed

Online Theses and Dissertations

Oxidative stress has been associated in the pathogenesis of numerous diseases such as neurodegenerative disorders, ischemia, and cancer. The brain is susceptible to oxidative stress due to its high content of peroxidizable unsaturated fatty acids, high consumption of oxygen per unit weight, high levels of free radicals, and comparatively low levels of antioxidant defense systems. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) can react with biomolecules such as proteins, lipids, carbohydrates, DNA, and RNA, which can lead to oxidative damage, cellular dysfunction, and can ultimately cause cell death. Down syndrome (DS) is the most common form of chromosomal …

Characterization Of Pregnancy Induced Cardiac Hypertrophy In Rats, Patricia Holden

Online Theses and Dissertations

During pregnancy, an increase in blood volume occurs to compensate for fetal development, resulting in cardiac hypertrophy. For the majority of women, this physiological cardiac hypertrophy resolves following pregnancy. Prolonged cardiac hypertrophy can lead to heart failure. We propose that by studying the biochemical mechanisms that mediate healthy cardiac remodeling associated with pregnancy, we will gain a better understanding of mechanisms involved in pathological cardiac hypertrophy. To determine the biochemical changes that occur during pregnancy induced cardiac remodeling, we subjected rats to timed matings and collected morphological and biochemical data from not pregnant, 19 days pregnant, and 24 hours postpartum. …

Altered Self-Assembly And Apatite Binding Of Amelogenin Induced By N-Terminal Proline Mutation, Li Zhu, Vuk Uskoković, Thuan Le, Pamela Denbesten, Yulei Huang, Stefan Habelitz, Wu Li

Pharmacy Faculty Articles and Research

Objective—A single Pro-70 to Thr (p.P70T) mutation of amelogenin is known to result in hypomineralized amelogenesis imperfecta (AI). This study aims to test the hypothesis that the given mutation affects the self-assembly of amelogenin molecules and impairs their ability to conduct the growth of apatite crystals.

Design—Recombinant human full-length wild-type (rh174) and p.P70T mutated amelogenins were analyzed using dynamic light scattering (DLS), protein quantification assay and atomic force microscopy (AFM) before and after the binding of amelogenins to hydroxyapatite crystals. The crystal growth modulated by both amelogenins in a dynamic titration system was observed using AFM.

Results—As …

Targeted Identification Of Metastasis-Associated Cell-Surface Sialoglycoproteins In Prostate Cancer, Lifang Yang, Julius O. Nyalwidhe, Sigi Guo, Richard R. Drake, O. John Semmes

Bioelectrics Publications

Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC₄ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins …

An Apoptosis Targeted Stimulus With Nanosecond Pulsed Electric Fields (Nspefs) In E4 Squamous Cell Carcinoma, Wei Ren, Stephen J. Beebe

Bioelectrics Publications

Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was [95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels. …

The Proteomic Response Of Sea Squirts (Genus Ciona Congeners) To Heat Stress: Evidence For Differential Thermal Sensitivities, Loredana Serafini

Master's Theses

The sea squirts Ciona intestinalis and C. savignyi have disparate distribution patterns, which may result from differences in their thermal tolerance limits. Because C. intestinalis, an almost cosmopolitan species, has a more widespread distribution, it is thought that it is better adapted to endure a wide range of temperatures. In order to compare the heat stress response between these two congeners, we studied global changes in protein expression, using a proteomics approach. To characterize the response to extreme heat stress, animals of both species were exposed to temperatures of 22°C, 25°C, and 28°C for 6 h, and then were …

Roles Of The 15-Kda Selenoprotein (Sep15) In Redox Homeostasis And Cataract Development Revealed By The Analysis Of Sep 15 Knockout Mice, Marina V. Kasaikina, Dmitri E. Fomenko, Vyacheslav M. Labunskyy, Salil A. Lachke, Wenya Qiu, Juliet A. Moncaster, Jie Zhang, Mark W. Wojnarowicz, Sathish Kumar Natarajan, Mikalai I. Malinouski, Ulrich Schweizer, Petra A. Tsuji, Bradley A. Carlson, Richard L. Maas, Marjorie F. Lou, Lee E. Goldstein, Dolph L. Hatfield, Vadim N. Gladyshev

School of Veterinary and Biomedical Sciences: Faculty Publications

The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteinerich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable …