Biochemistry, Biophysics, and Structural Biology Commons^™

Measurements Of Reactive Trace Gases And Variable O-3 Formation Rates In Some South Carolina Biomass Burning Plumes, S. K. Akagi, Robert J. Yokelson, I. R. Burling, S. Meinardi, Isobel J. Simpson, Donald R. Blake, G. R. Mcmeeking, A. Sullivan, T. Lee, S. Kreidenweis, S. P. Urbanski, J. Reardon, David W. T. Griffith, T. J. Johnson, D. R. Weise

Chemistry and Biochemistry Faculty Publications

In October-November 2011 we measured trace gas emission factors from seven prescribed fires in South Carolina (SC), US, using two Fourier transform infrared spectrometer (FTIR) systems and whole air sampling (WAS) into canisters followed by gas-chromatographic analysis. A total of 97 trace gas species were quantified from both airborne and ground-based sampling platforms, making this one of the most detailed field studies of fire emissions to date. The measurements include the first emission factors for a suite of monoterpenes produced by heating vegetative fuels during field fires. The first quantitative FTIR observations of limonene in smoke are reported along with …

Histone Deacetylase 1: Mutagenesis And Small Molecule Studies, Magdalene Wambua

Wayne State University Dissertations

Histone deacetylase 1 (HDAC1) has been linked to cell growth and cell cycle regulation, which makes it a widely recognized target for anticancer drugs. The 14 Å channel of Class 1 HDAC isotypes has long being hypothesized to be the exit cavity for acetate following deacetylation. The amino acids lining this cavity are very similar among the HDAC isoforms, suggesting the role of the cavity is relevant to all HDACs proteins Importanly, HDAC1 selective inhibitors designed to fit the 14Å channel have been designed. To understand the importance of the 14Å channel to HDAC1 activity, we used an alanine scan …

The Role Of Cardiolipin In Iron Homeostasis And Glutathione Metabolism, Vinay A. Patil

Wayne State University Dissertations

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. Inside the mitochondria, CL is a critical target of mitochondrial generated reactive oxygen species (ROS) and regulates signaling events related to apoptosis and aging. CL deficiency causes perturbation of signaling pathways outside the mitochondria, including the PKC-Slt2 cell integrity pathway and the high osmolarity glycerol (HOG) pathway, and is a key player in the cross-talk between the mitochondria and the vacuole. The importance of CL in human health is underscored by the observation that perturbation of CL …

Synthesis And Application Of Atp Analogs For Phosphorylation-Dependent Kinase-Substrate Crosslinking, Satish Kumar Garre Venkata Raghavendra

Wayne State University Dissertations

Phosphorylation is an important post-translational modification that plays a key role in a variety of signaling cascades and cellular functions. Kinases phosphorylate protein substrates in a highly regulated manner and are promiscuous. Understanding kinase-substrate specificity has been challenging and there is a need for new chemical tools. To this end we developed -phosphate modified ATP photocrosslinking analogs ATP-ArN3 and ATP-BP, that crosslink substrate and kinase in a phosphorylation dependent manner. We have successfully demonstrated that ATP-ArN3 and ATP-BP can be used with natural kinase and substrates using cell lysates in vitro. We used our approach to identify novel kinases of …

Defending Your Dna: Combating Threats Both Foreign And Domestic, James Mcisaac

Undergraduate Review

We are under constant assault from forces capable of damaging our DNA. The genetic code of DNA is made up of four nucleotides: adenine (A) which bonds with thymine (T) and guanine (G) which bonds with cytosine (C). If something happens to upset this normal pairing or the nucleotides themselves, our body must spring to action and respond to the damage. When damage to nucleotides prevents the normal replication machinery from doing its job, enzymes like the Y-family polymerases are called in. A special mechanism allows them to identify damage and insert the correct nucleotide or bypass the lesion to …

Studies Of Metabolite–Protein Interactions: A Review, Ryan Matsuda, Cong Bi, Jeanethe Anguizola, Matthew Sobansky, Elliott Rodriguez, John Vargas Badilla, Xiwei Zheng, Benjamin Hage, David S. Hage

David Hage Publications

The study of metabolomics can provide valuable information about biochemical pathways and processes at the molecular level. There have been many reports that have examined the structure, identity and concentrations of metabolites in biological systems. However, the binding of metabolites with proteins is also of growing interest. This review examines past reports that have looked at the binding of various types of metabolites with proteins. An overview of the techniques that have been used to characterize and study metabolite–protein binding is first provided. This is followed by examples of studies that have investigated the binding of hormones, fatty acids, drugs …

Bioinformatic And Functional Analysis Of 1-Aminocyclopropane-1-Carboxylate (Acc)- Deaminase Homologues In Strains Of Sinorhizobium, Alyssa L. Renn

Departmental Honors Projects

Nitrogen fixing bacteria like Sinorhiziobia have the ability to convert nitrogen gas into ammonia, allowing its incorporation into proteins and other biological molecules. These organisms form a symbiosis with legume plants such as peas, soybeans, and alfalfa. The symbiotic relationship between Sinorhizobia and legumes requires of the function of many proteins; one is 1- aminocyclopropane-1-carboxylic acid (ACC)-deaminase. Bacteria containing ACC-deaminase enzymes are more effective in nodulation due to their ability to lower the concentration of ethylene and bypass the plant defense systems. Upon examination of the complete DNA sequence of forty-eight Sinorhiziobia strains from different geographical locations, eighteen sequences similar …

Proline Mechanisms Of Stress Survival, Xinwen Liang, Lu Zhang, Sathish Kumar Natarajan, Donald F. Becker

Department of Biochemistry: Faculty Publications

Significance: The imino acid proline is utilized by different organisms to offset cellular imbalances caused by environmental stress. The wide use in nature of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. Understanding the mechanisms by which proline enhances abiotic/biotic stress response will facilitate agricultural crop research and improve human health. Recent Advances: It is now recognized that proline metabolism propels cellular signaling processes that promote cellular apoptosis or survival. Studies have shown that proline metabolism influences signaling pathways by increasing reactive oxygen species (ROS) formation in the mitochondria …

Comparative Metabolic Profiling Of Haberlea Rhodopensis, Thellungiella Halophyla, And Arabidopsis Thaliana Exposed To Low Temperature, Maria Benina, Toshihiro Obata, Nikolay Mehterov, Ivan Ivanov, Veselin Petrov, Valentina Toneva, Alisdair R. Fernie, Tsanko S. Gechev

Department of Biochemistry: Faculty Publications

Haberlea rhodopensis is a resurrection species with extreme resistance to drought stress and desiccation but also with ability to withstand low temperatures and freezing stress. In order to identify biochemical strategies which contribute to Haberlea's remarkable stress tolerance, the metabolic reconfiguration of H. rhodopensis during low temperature (4^◦C) and subsequent return to optimal temperatures (21^◦C) was investigated and compared with that of the stress tolerant Thellungiella halophyla and the stress sensitive Arabidopsis thaliana. Metabolic analysis by GC-MS revealed intrinsic differences in the metabolite levels of the three species even at 21^◦C. H. rhodopensis …

Acanthamoeba Castellanii Of The T4 Genotype Is A Potential Environmental Host For Enterobacter Aerogenes And Aeromonas Hydrophila, Farzana Abubakar Yousuf, Ruqaiyyah Siddiqui, Naveed Ahmed Khan

Department of Biological & Biomedical Sciences

Background: Acanthamoeba can interact with a wide range of microorganisms such as viruses, algae, yeasts, protists and bacteria including Legionella pneumophila, Pseudomonas aeruginosa, Vibrio cholerae, Helicobacter pylori, Listeria monocytogenes, Mycobacterium spp., and Escherichia coli. In this capacity, Acanthamoeba has been suggested as a vector in the transmission of bacterial pathogens to the susceptible hosts.
Methods: Here, we used a keratitis isolate of A. castellanii of the T4 genotype and studied its interactions with two bacterial genera which have not been tested before, Enterobacter aerogenes, and Aeromonas hydrophila, as well as E. coli. Assays were performed to determine bacterial association with …

Development And Optimization Of The First High Throughput In Vitro Fret Assay To Characterize The Saccharomyces Cerevisiae Gpi-T, Sandamali Amarasingha Ekanayaka

Wayne State University Dissertations

DEVELOPMENT AND OPTIMIZATION OF AN IN VITRO FRET ASSAY TO CHARACTERIZE THE SACCHAROMYCES CEREVISIAE GPI TRANSAMIDASE

By

SANDAMALI AMARASINGHA EKANAYAKA

December 2013

Advisor: Dr. Tamara L. Hendrickson

Major: Biochemistry

Degree: Doctor of Philosophy

The enzyme glycosylphosphatidylinositol transamidase (GPI-T) mediates the attachment of a glycosylphosphatidylinositol (GPI) anchor to the C-terminus of specific proteins to produce GPI anchored proteins. This post-translational modification is essential for viability of eukaryotic organisms. However, very little is known about GPI-T and its catalytic activity. Thus, the research described in this abstract was conducted to develop an in vitro assay to monitor GPI-T. A high-throughput assay for …

Characterization Of A Novel Fusion Protein Of Ipab And Ipad Of Shigella And Its Potential As A Pan-Shigella Vaccine, F. J. Martinez-Becerra, X. Chen, Nicholas E. Dickenson, S. P. Choudhari, K. Harrison, J. D. Clements, W. D. Picking, L. L. Van De Verg, R. I. Walker, W. L. Picking

Chemistry and Biochemistry Faculty Publications

Shigellosis is an important disease in the developing world, where about 90 million people become infected with Shigella spp. each year. We previously demonstrated that the type three secretion apparatus (T3SA) proteins IpaB and IpaD are protective antigens in the mouse lethal pulmonary model. In order to simplify vaccine formulation and process development, we have evaluated a vaccine design that incorporates both of these previously tested Shigella antigens into a single polypeptide chain. To determine if this fusion protein (DB fusion) retains the antigenic and protective capacities of IpaB and IpaD, we immunized mice with the DB fusion and compared …

Acyl-Lipid Metabolism, Younghua Li-Beisson, Basil Shorrosh, Fred Beisson, Mats X. Andersson, Vincent Arondel, Philip D. Bates, Sébastien Baud, David Bird, Allan Debono, Timothy P. Durrett, Rochus B. Franke, Ian A. Graham, Kenta Katayama, Amélie A. Kelly, Tony Larson, Jonathan E. Markham, Martine Miquel, Isabel Molina, Ikuo Nishida, Owen Rowland, Lacey Samuels, Katherine M. Schmid, Hajime Wada, Ruth Welti, Changcheng Xu, Rémi Zallot, John Ohlrogge

Scholarship and Professional Work - LAS

Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes …

The Magnitude And Mechanism Of Charge Enhancement Of Ch∙∙O H-Bonds, U. Adhikari, Steve Scheiner

Chemistry and Biochemistry Faculty Publications

Quantum calculations find that neutral methylamines and thioethers form complexes, with N-methylacetamide (NMA) as proton acceptor, with binding energies of 2–5 kcal/mol. This interaction is magnified by a factor of 4–9, bringing the binding energy up to as much as 20 kcal/mol, when a CH3+ group is added to the proton donor. Complexes prefer trifurcated arrangements, wherein three separate methyl groups donate a proton to the O acceptor. Binding energies lessen when the systems are immersed in solvents of increasing polarity, but the ionic complexes retain their favored status even in water. The binding energy is reduced when the methyl …

Computational Approaches To Anti-Toxin Therapies And Biomarker Identification, Rebecca Jane Swett

Wayne State University Dissertations

This work describes the fundamental study of two bacterial toxins with computational methods, the rational design of a potent inhibitor using molecular dynamics, as well as the development of two bioinformatic methods for mining genomic data.

Clostridium difficile is an opportunistic bacillus which produces two large glucosylating toxins. These toxins, TcdA and TcdB cause severe intestinal damage. As Clostridium difficile harbors considerable antibiotic resistance, one treatment strategy is to prevent the tissue damage that the toxins cause. The catalytic glucosyltransferase domain of TcdA and TcdB was studied using molecular dynamics in the presence of both a protein-protein binding partner and …

Phase Composition Control Of Calcium Phosphate Nanoparticles For Tunable Drug Delivery Kinetics And Treatment Of Osteomyelitis. Part 2: Antibacterial And Osteoblastic Response, Vuk Uskoković, Tejal A. Dasai

Pharmacy Faculty Articles and Research

Osteomyelitis has been traditionally treated by the combination of long-term antibiotic therapies and surgical removal of diseased tissue. The multifunctional material was developed in this study with the aim to improve this therapeutic approach by: (a) enabling locally delivered and sustained release of antibiotics at a tunable rate, so as to eliminate the need for repetitive administration of systemically distributed antibiotics; and (b) controllably dissolving itself, so as to promote natural remineralization of the portion of bone lost to disease. We report hereby on the effect of the previously synthesized calcium phosphates (CAPs) with tunable solubilities and drug release time …

Revisiting The Fundamentals In The Design And Control Of Nanoparticulate Colloids In The Frame Of Soft Chemistry, Vuk Uskoković

Pharmacy Faculty Articles and Research

This review presents thoughts on some of the fundamental features of conceptual models applied in the design of fine particles in the frames of colloid and soft chemistry. A special emphasis is placed on the limitations of these models, an acknowledgment of which is vital in improving their intricacy and effectiveness in predicting the outcomes of the corresponding experimental settings. Thermodynamics of self-assembly phenomena illustrated on the examples of protein assembly and micellization is analyzed in relation to the previously elaborated thesis that each self-assembly in reality presents a co-assembly, since it implies a mutual reorganization of the assembling system …

Investigating Therapeutic Options For Lafora Disease Using Structural Biology And Translational Methods, Amanda R. Sherwood

Theses and Dissertations--Molecular and Cellular Biochemistry

Lafora disease (LD) is a rare yet invariably fatal form of epilepsy characterized by progressive degeneration of the central nervous and motor systems and accumulation of insoluble glucans within cells. LD results from mutation of either the phosphatase laforin, an enzyme that dephosphorylates cellular glycogen, or the E3 ubiquitin ligase malin, the binding partner of laforin. Currently, there are no therapeutic options for LD, or reported methods by which the specific activity of glucan phosphatases such as laforin can be easily measured. To facilitate our translational studies, we developed an assay with which the glucan phosphatase activity of laforin as …

Valence-Length Correlations For Chemical Bonds From Atomic Orbital Exponents, F. D. Hardcastle

Journal of the Arkansas Academy of Science

Pauling’s empirical bond valence-length correlation has proven valuable because it offers a quick and convenient way of checking and evaluating molecular structures and determining oxidation states from measured bond lengths. In this study, a simplified quantum-mechanical approach was used to derive Pauling’s empirical bond valence-length relationship by considering overlap of hydrogen-like orbitals. An expression for the b “empirical” fitting parameter was derived in terms of atomic-orbital exponents. A new set of orbital exponents is presented using published atomic/covalent radii and a continuous function for the effective principal quantum. The b parameters calculated from the orbital exponents are consistent with bond …

Synthesis, Characterization, And Properties Of Mononuclear And Dinuclear Ruthenium(Ii) Complexes Containing Phenanthroline And Chlorophenanthroline, Anwar A. Bhuiyan, X. Du

Journal of the Arkansas Academy of Science

The study of photophysical and photochemical properties of ruthenium complexes is of great interest for fundamental practical reasons. Ruthenium complexes have been investigated for use in artificial photosynthesis. This paper deals with the synthesis and spectroscopic investigation of custom-designed ruthenium complexes containing phenanthroline and chloro-phenanthroline ligands. These complexes maybe useful for biological electron-transfer studies. The heteroleptic ruthenium monomer complex Ru(phen)2(Cl-phen) (where phen = 1,10-phenanthroline and Cl-phen=5-chloro-1,10-phenanthroline) was prepared in a two-step procedure previously developed in our laboratory. This monomer complex was used to prepare the ruthenium homometallic dimer complex, (phen)2Ru(phen-phen)Ru(phen)2, by utilizing the Ni-catalyzed coupling reaction. Both complexes were purified …

Primary Pathways Of Intracellular Ca2+ Mobilization By Nanosecond Pulsed Electric Field, Iurii Semenov, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Permeabilization of cell membranous structures by nanosecond pulsed electric field (nsPEF) triggers transient rise of cytosolic Ca²⁺ concentration ([Ca²⁺]_i), which determines multifarious downstream effects. By using fast ratiometric Ca²⁺ imaging with Fura-2, we quantified the external Ca²⁺ uptake, compared it with Ca²⁺ release from the endoplasmic reticulum (ER), and analyzed the interplay of these processes. We utilized CHO cells which lack voltage-gated Ca²⁺ channels, so that the nsPEF-induced [Ca²⁺]_i changes could be attributed primarily to electroporation. We found that a single 60-ns pulse caused fast [Ca2+]_i increase …

Recruitment Of The Intracellular Ca2+ By Ultrashort Electric Stimuli: The Impact Of Pulse Duration, Iurii Semenov, Shu Xiao, Olga N. Pakhomova, Andrei G. Pakhomov

Bioelectrics Publications

Nanosecond-duration electric stimuli are distinguished by the ability to permeabilize intracellular membranes and recruit Ca²⁺ from intracellular stores. We quantified this effect in non-excitable cells (CHO) using ratiometric Ca²⁺ imaging with Fura-2. In a Ca²⁺-free medium, 10-, 60-, and 300-ns stimuli evoked Ca2⁺ transients by mobilization of Ca²⁺ from the endoplasmic reticulum. With 2 mM external Ca²⁺, the transients included both extra- and intracellular components. The recruitment of intracellular Ca²⁺ increased as the stimulus duration decreased. At the threshold of 200–300 nM, the transients were amplified by calcium-induced calcium release. We …

The Hyaluronan Receptor For Endocytosis (Hare) Activates Nf-Κb-Mediated Gene Expression In Response To 40–400-Kda, But Not Smaller Or Larger, Hyaluronans, Madhu S. Pandey, Bruce A. Baggenstoss, Jennifer Washburn, Edward N. Harris, Paul H. Weigel

Department of Biochemistry: Faculty Publications

Background: HARE mediates systemic clearance of hyaluronan (HA), which turns over continuously in tissues.

Results: HARE uptake of 40–400-kDa, but not larger or smaller, HA stimulated NF-κB activation.

Conclusion: HA-HARE signal complexes activate NF-κB and gene transcription only with optimally sized HA.

Significance: HARE responsiveness to a narrow size range ofHAdegradation products may be a sensing system to detect tissue ECM stress.

Investigating The Effects Of Bag3 Knockdown On The Cytotoxicity Of The Anticancer Drug Laromustine, Kayla Gross

Honors Theses

Laromustine is a sulfonylhydrazine anticancer prodrug whose cytotoxicity results from the formation of interstrand cross-links caused by the synergistic action of co- generated 2-chloroethylating and carbamoylating species. The cytotoxic activities of Laromustine involve the induction of apoptosis. Described herein is an investigation into this drug’s effects on apoptotic gene expression in HL-60 cells using qRT-PCR. Significant changes in the expression levels of 13 genes were observed, most dramatically in the upregulation of the bcl2-associated athanogene 3 (BAG3) gene. Given the pro-survival role of BAG3 in the cell, this investigation sought to decrease BAG3 mRNA levels in HL-60 cells using transient …

Relationship Of Cross-Linking Potential To Mechanism Of Cell Death, Adam N. Spierer

Honors Theses

Mechlorethamine (HN2), a nitrogen derivative of mustard gas, was the first synthetic anti-tumor chemotherapeutic because it forms covalent cross-links between strands of duplex DNA. HN2 represents a class of bifunctional alkylating agents that are both chemotherapeutic and carcinogenic: diepoxybutane (DEB), the active form of the pro-drug treosulfan, and epichlorohydrin (ECH), a structural hybrid of HN2 and DEB, also form covalent cross-links between DNA. While HN2 and DEB are clinically used as anti-tumor chemotherapeutics, ECH is a structural hybrid of these two compounds not used in a clinical setting. Accordingly, we aimed to understand the relationship between the cross-linking potential of …

Intercalibraton In Chemical Oceanography-- Getting The Right Number, Gregory A. Cutter

OES Faculty Publications

Intercalibration has a strict metrological definition, but in brief, it's an open sharing of methods and results between laboratories to achieve the most accurate data with the fewest random and systematic errors. In the field of chemical oceanography where concentrations of many constituents can be in the nano- to picomolar range, the salt water matrix can be difficult to analyze, and knowing the exact concentrations, or even chemical forms, of biologically required elements is essential, intercalibration is a very relevant and needed tool. Implementing it is not simple because errors can occur at any step in the process of taking …

Using Ammonium Pore Water Profiles To Assess Stoichiometry Of Deep Remineralization Processes In Methanogenic Continental Margin Sediments, David J. Burdige, Tomoko Komada

OES Faculty Publications

In many continental margin Sediments, a deep reaction zone exists which is separated from remineralization processes near the sediment surface. Here, methane diffuses upward to a depth where it is oxidized by downwardly diffusing sulfate. However, the methane sources that drive this anaerobic oxidation of methane (AOM) in the sulfate-methane transition zone (SMT) may vary among sites. In particular, these sources can be thought of as either (i) "internal" sources from in situ methanogenesis (regardless of where it occurs in the sediment column) that are ultimately coupled to organic matter deposition and burial, or (ii) "external" sources such as hydrocarbon …

Distributions Of Particulate Heme B In The Atlantic And Southern Oceans- Implications For Electron Transport In Phytoplankton, Martha Gledhill, Eric P. Achterberg, David J. Honey, Maria C. Nielsdottir, Micha J.A. Rijkenberg

OES Faculty Publications

Concentrations of heme b, the iron-containing component of b-type hemoproteins, ranged from b concentrations were enhanced in the photic zone and decreased with depth. Heme b concentrations correlated positively with chlorophyll a (chl a) in the TNA (r=0.41, pb did not correlate with chl a in the IB or SS. In the IB and SS, stations with high-chlorophyll and low-nutrient (Fe and/or Si) concentrations exhibited low heme bconcentrations relative to particulate organic carbon (< 0.1 μmolmol^-1, and high chl a:heme b ratios (> 500). High chl a:heme b ratios resulted from relative decreases in …

Membrane Surface Charges Attracted Protons Are Not Relevant To Proton Motive Force, James Weifu Lee

Chemistry & Biochemistry Faculty Publications

[First Paragraph] Only a half-century ago, British scientist Peter Mitchell proposed the conceptual scheme known as "chemiosmotic theory" that identified the role of proton coupling in the formation of ATP in cells [1,2]. Mitchell's delocalized proton-coupling chemiosmotic theory was initially quite controversial. But it would win for him the Nobel Prize in Chemistry in 1978. However, questions about Mitchell's chemiosmotic hypothesis have never gone away and a number of credible studies indicated the full extent of Mitchell's idea didn't always hold up to experience.

Solid-Phase Organic Matter Reduction Regulates Anaerobic Decomposition In Bog Soil, Jason K. Keller, Kimberly K. Takagi

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Peatlands store globally significant amounts of carbon and are important sources of the greenhouse gas methane (CH4) to the atmosphere. However, for reasons which are not well understood, many peatland soils produce smaller amounts of CH4 than theoretically predicted, and carbon dioxide (CO2) produced during anaerobic decomposition in peatland soils cannot be accounted for by commonly measured microbial processes. Here we show that the reduction of solid-phase organic matter (i.e., humic substances) suppresses CH4 production in a bog soil and can be responsible for 33–61% of the total carbon mineralization in this soil. These results demonstrate that the reduction of …