Other Biochemistry, Biophysics, and Structural Biology Commons^™

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

Colin L. Weeks

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 ...

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 Becker, Thomas G. Spiro, James Penner-Hahn, Ruma Banerjee

Colin L. Weeks

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 ...

Dietary Phytochemicals As Inhibitors Of Primary Amine Oxidase, Padraig Shanahan, Jeffrey O'Sullivan, Keith F. Tipton, Gemma Kinsella, Barry J. Ryan, Gary T. Henehan

Articles

Phytochemicals such as methylxanthines, catechins and polyphenols show health benefits in a range of diseases although their mechanism of action is not fully understood. Primary Amine Oxidase (PrAO) is widely recognised as a therapeutic drug target for the treatment of inflammatory, vascular and neurodegenerative diseases. Previous work in our laboratories showed that caffeine inhibited bovine PrAO activity with a Ki of 1.0mM. In the present study we examined a range of methylxanthines and catechins as inhibitors of bovine PrAO. The methylxanthines tested were caffeine, paraxanthine, theophylline, theobromine and 7-methylxanthine. Of these, only theobromine was an inhibitor with an IC50 ...

Metalloproteases Of The Inner Mitochondrial Membrane, Roman M. Levytskyy, Iryna Bohovych, Oleh Khalimonchuk

Biochemistry -- Faculty Publications

The inner mitochondrial membrane (IM) is among most protein-rich cellular compartments. The metastable IM sub-proteome where the concentration of proteins is approaching oversaturation creates a challenging protein folding environment with high probability for protein malfunction or aggregation. Failure to maintain protein homeostasis in such a setting can impair functional integrity of the mitochondria and drive clinical manifestations. The IM is equipped with a series of highly conserved, proteolytic complexes dedicated to the maintenance of normal protein homeostasis within this mitochondrial sub-compartment. Particularly important is a group of membrane-anchored metallopeptidases commonly known as m-AAA and i-AAA proteases, and the ATP-independent Oma1 ...

The Role Of Interactions Of Long Non-Coding Rnas And Heterogeneous Nuclear Ribonucleoproteins In Regulating Cellular Functions, Xinghui Sun, Mohamed Sham Shihabudeen Haider Ali, Matthew Moran

Biochemistry -- Faculty Publications

Long non-coding RNAs (lncRNAs) are emerging as critical regulators of various biological processes and human diseases. The mechanisms of action involve their interactions with proteins, RNA and genomic DNA. Most lncRNAs display strong nuclear localization. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of RNA-binding proteins that are important for multiple aspects of nucleic acid metabolism. hnRNPs are also predominantly expressed in the nucleus. This review discusses the interactions of lncRNAs and hnRNPs in regulating gene expression at transcriptional and post-transcriptional levels or by changing genomic structure, highlighting their involvements in glucose and lipid metabolism, immune response, DNA damage response ...

Elucidating Mechanisms Of Protein Aggregation In Alzheimer’S Disease Using Antibody-Based Strategies., Benjamin A. Colvin

Dissertations

Alzheimer’s Disease (AD) is a devastating neurodegenerative disorder. There are two characteristic histopathological hallmarks in the brain: senile plaques and neurofibrillary tangles, composed of insoluble aggregates of the amyloids Amyloid-β (Aβ) and tau protein, respectively. These diagnostic markers, though distinctive, are not apparent effectors of AD pathology. Evidence has mounted suggesting smaller soluble aggregates (oligomers) of Aβ or tau are the true drivers of disease progression. This dissertation presents several amyloid biophysics projects. Aggregate biophysical parameters such as weight, shape, and conformation were measured using a range of methodologies, including Multiangle Light Scattering, Dynamic Light Scattering, UV-Circular Dichroism, UV-Fluorescence ...

Correction For Sandai Et Al., The Evolutionary Rewiring Of Ubiquitination Targets Has Reprogrammed The Regulation Of Carbon Assimilation In The Pathogenic Yeast Candida Albicans, Doblin Sandai, Zhikang Yin, Laura Selway, David Stead, Janet Walker, Michelle D. Leach, Iryna Bohovych, Iuliana V. Ene, Stavroula Kastora, Susan Budge, Carol A. Munro, Frank C. Odds, Neil A.R. Gow, Alistair J.P. Brown

Janet Walker

No abstract provided.

The Evolutionary Rewiring Of Ubiquitination Targets Has Reprogrammed The Regulation Of Carbon Assimilation In The Pathogenic Yeast Candida Albicans, Doblin Sandai, Zhikang Yin, Laura Selway, David Stead, Janet Walker, Michelle D. Leach, Iryna Bohovych, Iuliana V. Ene, Stavroula Kastora, Susan Budge, Carol A. Munro, Frank C. Odds, Neil A.R. Gow, Alistair J.P. Brown

Janet Walker

Microbes must assimilate carbon to grow and colonize their niches. Transcript profiling has suggested that Candida albicans, a major pathogen of humans, regulates its carbon assimilation in an analogous fashion to the model yeast Saccharomyces cerevisiae, repressing metabolic pathways required for the use of alterative nonpreferred carbon sources when sugars are available. However, we show that there is significant dislocation between the proteome and transcriptome in C. albicans. Glucose triggers the degradation of the ICL1 and PCK1 transcripts in C. albicans, yet isocitrate lyase (Icl1) and phosphoenolpyruvate carboxykinase (Pck1) are stable and are retained. Indeed, numerous enzymes required for the ...

Approaching Undergraduate Research With Students Who Are Deaf And Hard-Of-Hearing, Austin U. Gehret, Jessica W. Trussell, Lea V. Michel

Journal of Science Education for Students with Disabilities

An undergraduate research experience can provide a unique opportunity for students to learn and grow as scientists; when positive, this experience is often transformative and motivates students to pursue science, technology, engineering and mathematics (STEM) graduate degrees or careers. Conversely, negative research experiences can sour a student’s opinion of research, propagate misconceptions of graduate school, and lead to attrition from STEM fields. Negative research experiences can be equally devastating for faculty mentors and may result in reluctance to mentor future research students. Using a mentoring approach that has traditionally translated to positive research experiences for hearing students may not ...

Inquiry Into Perilipin-5a Expression In Triacylglycerol Rich Vs Normal Fed Mouse Tissue, Kobi Agyepong

Honors Thesis Projects

The steep rise in both childhood and adult obesity over the past three decades has moved to the forefront of public consciousness in recent years. This development has generated a marked increase in general health awareness and lifestyle changes for a vast number of individuals, most notably in the form of increased physical activity and diet alterations. The latter point is especially salient in a biochemical context, because of the myriad factors that can result in “fat accumulation”. Chief among these factors is the Perilipin 5A gene, (known as PLIN5A) which encodes the protein Perilipin 5A of the Perilipin family ...

The Effects Of Intrauterine Growth Restriction (Iugr) On The Pulmonary Surfactant And Lung Injury, Reza Khazaee

Western Research Forum

The Effects of Intrauterine Growth Restriction (IUGR) on the Pulmonary Surfactant and Lung Injury

Khazaee R¹, McCaig LA², Hardy D¹, Yamashita CM², Veldhuizen, RAW²

Department of Physiology and Pharmacology¹, Western University, London, ON, Canada. Lawson Health Research Institute², London, ON, Canada

Background: Acute respiratory distress syndrome (ARDS) is defined as severe lung dysfunction that occurs after an insult to the lung such as an infection. The lung dysfunction in ARDS is due to alterations to surfactant, a lipid-protein mixture coats the inside of the lung and maintains the lungs’ ability to expand easily ...

Analysis Of Resistant Starches In Rat Cecal Contents Using Fourier Transform Infrared Photoacoustic Spectroscopy, Timothy J. Anderson, Yongfeng Ai, Roger W. Jones, Robert S. Houk, Jay-Lin Jane, Yinsheng Zhao, Diane F. Birt, John F. Mcclelland

Roger W. Jones

Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS) qualitatively and quantitatively measured resistant starch (RS) in rat cecal contents. Fisher 344 rats were fed diets of 55% (w/w, dry basis) starch for 8 weeks. Cecal contents were collected from sacrificed rats. A corn starch control was compared against three RS diets. The RS diets were high-amylose corn starch (HA7), HA7 chemically modified with octenyl succinic anhydride, and stearic-acid-complexed HA7 starch. To calibrate the FTIR-PAS analysis, samples from each diet were analyzed using an enzymatic assay. A partial least-squares cross-validation plot generated from the enzymatic assay and FTIR-PAS spectral results for starch ...

An Assessment Of Potential False Positive E.Coli Pyroprints In The Cplop Database, Skyler A. Gordon

Master's Theses and Project Reports

The genetic information found in each species of organism is unique, and can be used as a tool to differentiate at the molecular level. This has caused rapid genotyping methods to become the cornerstone of a new area of research dependent on reading the genome as a form of identification. One of these specific identification methods, known as pyroprinting, relies on the small variation of DNA sequences within the same species to develop a unique, reproducible fingerprint. By simultaneously pyrosequencing multiple polymorphic loci within the ribosomal operons known as the intergenic transcribed spacers, a reproducible output is obtained, known as ...

Alpha-Synuclein: Insight Into The Hallmark Of Parkinson's Disease As A Target For Quantitative Molecular Diagnostics And Therapeutics, Baggio A. Evangelista

Honors in the Major Theses

Parkinson’s disease (PD) is the second-most common neurodegenerative disease after Alzheimer’s disease. With 500,000 individuals currently living with Parkinson’s and nearly 60,000 new cases diagnosed each year, this disease causes significant financial burden on the healthcare system - amassing to annual expenditures totaling 200 billion dollars; predicted to increase through 2050. The disease phenotype is characterized by a combination of a resting tremor, bradykinesia, muscular rigidity, and depression due to dopaminergic neuronal death in the midbrain. The cause of the neurotoxicity has been largely discussed, with strong evidence suggesting that the protein, alpha-Synuclein, is a key ...

Oxidative Stress, Metabolomics Profiling, And Mechanism Of Local Anesthetic Induced Cell Death In Yeast, Cory Honsinger Thomas Boone, Ryan A. Grove, Dana Adamcova, Javier Seravalli, Jiri Adamec

Biochemistry -- Faculty Publications

The World Health Organization designates lidocaine as an essential medicine in healthcare, greatly increasing the probability of human exposure. Its use has been associated with ROS generation and neurotoxicity. Physiological and metabolomic alterations, and genetics leading to the clinically observed adverse effects have not been temporally characterized. To study alterations that may lead to these undesirable effects, Saccharomyces cerevisiae grown on aerobic carbon sources to stationary phase was assessed over 6 h. Exposure of an LC₅₀ dose of lidocaine, increased mitochondrial depolarization and ROS/RNS generation assessed using JC-1, ROS/RNS specific probes, and FACS. Intracellular calcium also increased ...

Significant Enhancement Of Fatty Acid Composition In Seeds Of The Allohexaploid, Camelina Sativa, Using Crispr/Cas9 Gene Editing, Wen Zhi Jhang, Isabelle M. Henry, Peter G. Lynagh, Lucia Comai, Edgar B. Cahoon, Donald P. Weeks

Biochemistry -- Faculty Publications

The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were associated with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e. a decrease from ~16 ...

Toxoplasma Dj-1 Regulates Organelle Secretion By A Direct Interaction With Calcium-Dependent Protein Kinase 1, Matthew A. Child, Megan Garland

Biochemistry -- Faculty Publications

Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson’s disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-A resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation ...

Stability Of Peatland Carbon To Rising Temperatures, R. M. Wilson, A. M. Hopple, M. M. Tfaily, S. D. Sebestyen, C. W. Schadt, L. Pfeifer-Meister, Cassandra Medvedeff, K. J. Mcfarlane, J. E. Kostka, M. Kolton, R. K. Kolka, L. A. Kluber, Jason K. Keller, T. P. Guilderson, N. A. Griffiths, J. P. Chanton, S. D. Brigham, P. J. Hanson

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Peatlands contain one-third of soil carbon (C), mostly buried in deep, saturated anoxic zones (catotelm). The response of catotelm C to climate forcing is uncertain, because prior experiments have focused on surface warming. We show that deep peat heating of a 2 m-thick peat column results in an exponential increase in CH4 emissions. However, this response is due solely to surface processes and not degradation of catotelm peat. Incubations show that only the top 20–30 cm of peat from experimental plots have higher CH4 production rates at elevated temperatures. Radiocarbon analyses demonstrate that CH4 and CO2 are produced primarily ...

Expression Of Glycine-Rich Proteins Found In Salivary Glands Of The Lone Star Tick (Amblyomma Americanum) Using A Mammalian Cell Line, Annabelle Clark

Honors Theses

Ticks play an important ecological role as well as a growing role in human health and veterinary care. Ticks are hosts to a plethora of microbial pathogens that can be transferred during feeding to cause tick-borne diseases in humans and many animals. Ticks may in large part owe the success of the transfer of these pathogens between hosts to their complex saliva. The saliva secreted upon a tick’s attachment to a host serves the following, among other, functions: anti-hemostasis of the blood pool, preventing an inflammatory response at the bite site, and serving as a natural anti-microbial substance. An ...

Maintenance Of The Ph Gradient In The Gastric Mucus Layer., Owen Lewis

Annual Symposium on Biomathematics and Ecology: Education and Research

No abstract provided.