Biochemistry Commons^™

Stability Studies Of Membrane Proteins, Cui Ye

Theses and Dissertations--Chemistry

The World Health Organization has identified antimicrobial resistance as one of the top three threats to human health. Gram-negative bacteria such as Escherichia coli are intrinsically more resistant to antimicrobials. There are very few drugs either on the market or in the pharmaceutical pipeline targeting Gram-negative pathogens. Two mechanisms, the protection of the outer membrane and the active efflux by the multidrug transporters, play important roles in conferring multidrug resistance to Gram-negative bacteria. My work focuses on two main directions, each aligning with one of the known multidrug resistance mechanisms.

The first direction of my research is in the area …

Increase Of Basal Oxidative Stress Levels And Impairment Of Heme Oxygenase-1/Biliverdin Reductase Post-Translational Modification By The Defect Of Parkinson-Related Gene Of Pink1, Zhaoshu Zhang

Theses and Dissertations--Chemistry

Parkinson disease (PD) is the most common movement disorder and the second most common neurodegenerative disease. PINK1, PTEN-induced kinase 1, functions as a serine/threonine kinase as well as a protector of mitochondrial function. Mutations in PINK1 gene result in either mitochondria dysfunction or disruption of kinase signaling pathways involved in the pathogenesis of PD.

In this thesis, oxidative stress levels were examined in the brain of PINK1 knockout mice, and also how heme oxygenase-1 and biliverdin reductase are affected in brain of PINK1 knockout mice. In addition, posttranslational modifications are a way to control the behavior of proteins, so posttranslational …

The Differences Between Iron And Iron-Substituted Manganese Superoxide Dismutase With Respect To Hydrogen Peroxide Treatment, Jianing Wang

Theses and Dissertations--Chemistry

Iron-substituted manganese superoxide dismutase (Fe(Mn)SOD) was produced using an in vivo preparation method. It’s an inactive enzyme in catalyzing superoxide radical dismutation owing to the mis-incorporation of Fe in the active site evolved to use Mn. To investigate the possible toxicity of human Fe(Mn)SOD proposed by Yamakura, we studied the properties of Fe(Mn)SOD upon H₂O₂ treatment and compared to that of FeSOD. It’s found that the responses to H₂O₂ treatment were different, including the changes of optical spectra, variations of active site coordination and secondary structures. Fe³⁺ reduction was not observed in Fe(Mn)SOD …

Influence Of Media Composition On The Growth Rate Of Chlorella Vulgaris And Scenedesmus Acutus Utilized For Co2 Mitigation, Czarena L. Crofcheck, Xinyi E, Aubrey Shea, Michael D. Montross, Mark Crocker, Rodney Andrews

Biosystems and Agricultural Engineering Faculty Publications

Atmospheric carbon dioxide levels have increased since the industrial revolution due to increasing combustion of fossil fuels. One possible CO₂ mitigation strategy is the use of microalgae for mitigation of CO₂. This paper focuses on the influence of media composition on the growth rate of two microalgae strains, Chlorella vulgaris and Scenedesmus actus. A KNO₃ based medium was found to work better for Chlorella, while a urea based equivalent worked better for Scenedesmus. The urea based media investigated here resulted in growth similar to that found with previously reported KNO₃ based media. …

Bed Bugs Evolved Unique Adaptive Strategy To Resist Pyrethroid Insecticides, Fang Zhu, Hemant Gujar, Jennifer R. Gordon, Kenneth F. Haynes, Michael F. Potter, Subba R. Palli

Entomology Faculty Publications

Recent advances in genomic and post-genomic technologies have facilitated a genome-wide analysis of the insecticide resistance-associated genes in insects. Through bed bug, Cimex lectularius transcriptome analysis, we identified 14 molecular markers associated with pyrethroid resistance. Our studies revealed that most of the resistance-associated genes functioning in diverse mechanisms are expressed in the epidermal layer of the integument, which could prevent or slow down the toxin from reaching the target sites on nerve cells, where an additional layer of resistance (kdr) is possible. This strategy evolved in bed bugs is based on their unique morphological, physiological and behavioral characteristics …

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 …

Towards Elucidation Of A Viral Dna Packaging Motor, Chad T. Schwartz

Theses and Dissertations--Pharmacy

Previously, gp16, the ATPase protein of phi29 DNA packaging motor, was an enigma due to its tendency to form multiple oligomeric states. Recently we employed new methodologies to decipher both its stoichiometry and also the mechanism in which the protein functions to hydrolyze ATP and provide the driving force for DNA packaging. The oligomeric states were determined by biochemical and biophysical approaches. Contrary to many reported intriguing models of viral DNA packaging, it was found that phi29 DNA packaging motor permits the translocation of DNA unidirectionally and driven cooperatively by three rings of defined shape. The mechanism for the generation …

Amalgamation Of Nucleosides And Amino Acids In Antibiotic Biosynthesis, Sandra H. Barnard

Theses and Dissertations--Pharmacy

The rapid increase in antibiotic resistance demands the identification of novel antibiotics with novel targets. One potential antibacterial target is the biosynthesis of peptidoglycan cell wall, which is both ubiquitous and necessary for bacterial survival. Both the caprazamycin-related compounds A-90289 and muraminomicin, as well as the capuramycin-related compounds A-503083 and A-102395 are potent inhibitors of the translocase I enzyme, one of the key enzymes required for cell wall biosynthesis. The caprazamycin-related compounds contain a core nonproteinogen b-hydroxy-a-amino acid referred to as 5’-C-glycyluridine (GlyU). Residing within the biosynthetic gene clusters of the aforementioned compounds is a shared open reading …

Regulation Of 7-Dehydrocholesterol Reductase By Vitamin D3, Ling Zou

Theses and Dissertations--Pharmacy

7-Dehydrocholesterol (7-DHC) is the substrate of 7-dehydrocholesterol reductase (DHCR7) in the cholesterol synthesis pathway. Keratinocytes in human skin possess the enzymes necessary for cholesterol synthesis but are also responsible for vitamin D₃ synthesis from 7-DHC by exposure to UVB irradiation. It has been well established that DHCR7 is regulated by the SREBP pathway in the regulation of cholesterol synthesis, but little is known about the regulation of DHCR7 by the vitamin D pathway. In this study, the regulation of DHCR7 activity by vitamin D was explored. Treatment of adult human epidermal keratinocyte (HEKa) cells with vitamin D₃ resulted …

Critical Events In Human Metapneumovirus Infection: From Entry To Egress, Brent A. Hackett

Theses and Dissertations--Molecular and Cellular Biochemistry

Human metapneumovirus (HMPV) is a respiratory pathogen in Paramyxovirus family that demonstrates extremely high morbidity in the population, with most individuals having been infected by the age of five. Despite the prevalence of this negative-sense RNA virus in the population for decades, it was only identified in 2001. As such, there is currently no specific treatment for HMPV and the potentially severe consequences of infection for elderly and immunocompromised individuals and particularly infants make development of antivirals targeting HMPV of high significance. HMPV constitutes a quarter of all respiratory hospitalizations among infants, placing it second only to RSV, in addition …

The Disordered Regulation Of Calcineurin: How Calmodulin-Induced Regulatory Domain Structural Changes Lead To The Activation Of Calcineurin, Victoria B. Dunlap

Theses and Dissertations--Molecular and Cellular Biochemistry

Calcineurin (CaN) is a highly regulated Ser/Thr protein phosphatase that plays critical roles in learning and memory, cardiac development and function, and immune system activation. Alterations in CaN regulation contribute to multiple disease states such as Down syndrome, cardiac hypertrophy, Alzheimer’s disease, and autoimmune disease. In addition, CaN is the target of the immunosuppressant drugs FK506 and cyclosporin A. Despite its importance, CaN regulation is not well understood on a molecular level. Full CaN activation requires binding of calcium-loaded calmodulin (CaM), however little is known about how CaM binding releases CaN’s autoinhibitory domain from the active site. Previous work has …

Doxorubicin-Induced, Tnf-Α-Mediated Brain Oxidative Stress, Neurochemical Alterations, And Cognitive Decline: Insights Into Mechanisms Of Chemotherapy Induced Cognitive Impairment And Its Prevention, Jeriel T. Keeney

Theses and Dissertations--Chemistry

The works presented in this dissertation provide insights into the mechanisms of chemotherapy-induced cognitive impairment (CICI or “ChemoBrain”) and take steps toward outlining a preventive strategy. CICI is now widely recognized as a complication of cancer chemotherapy experienced by a large percentage of cancer survivors. Approximately fifty percent of existing FDA-approved anti-cancer drugs generate reactive oxygen species (ROS). Doxorubicin (Dox), a prototypical ROS-generating chemotherapeutic agent, produces the reactive superoxide radical anion (O₂^-•) in vivo. Dox treatment results in oxidation of plasma proteins, including ApoA-I, leading to TNF-α-mediated oxidative stress in plasma and brain. TNF-α elevation in brain …

Understanding Multidrug Resistance In Gram-Negative Bacteria -- A Study Of A Drug Efflux Pump Acrb And A Periplasmic Chaperone Sura, Meng Zhong

Theses and Dissertations--Chemistry

Multiple drug resistance (MDR) has been a severe issue in treatment and recovery from infection.Gram-negative bacteria intrinsically exhibit higher drug tolerance than Gram-positive microbes. In this thesis, two proteins involved in Gram-negative bacterial MDR were studied, AcrB and SurA.

Resistance-nodulation-cell division pump AcrAB-TolC is the major MDR efflux system in Gram-negative bacteria and efficiently extrudes a broad range of substances from the cells. To study subtle conformational changes of AcrB in vivo, a reporter platform was designed. Cysteine pairs were introduced into different regions in the periplasmic domain of the protein, and the extents of disulfide bond formation were …

Screening Synthetic Combinatorial Protein Libraries For Changes In Calmodulin Binding Specificity, Donald E. Dick

Kaleidoscope

No abstract provided.

Early Events Of Human Metapneumovirus Infection, Andres Chang

Theses and Dissertations--Molecular and Cellular Biochemistry

Human metapneumovirus (HMPV) is a worldwide respiratory pathogen that belongs to the paramyxovirus family of enveloped viruses and affects primarily the pediatric, geriatric, and immunocompromised populations. Despite its prevalence and importance to human health, no therapies are available against this pathogen. For paramyxoviruses, it is believed that infection starts by attachment of the virus to the surface of the cell through the viral attachment protein followed by fusion between the viral and cellular membranes, a process mediated by the fusion (F) protein at the plasma membrane and at neutral pH. Previous work showed that HMPV infection can occur in the …

Structural Basis Of Substrate Recognition In Thimet Oligopeptidase And Development Of Nanoparticles For Therapeutic Enzyme Delivery, Jonathan Mark Wagner

Theses and Dissertations--Molecular and Cellular Biochemistry

Neuropeptidases are responsible for degradation of signaling peptides in the central nervous system and periphery. Some neuropeptidases have also been shown to play a role as part of the cell’s hydrolytic machinery responsible for breaking down proteins and peptides into amino acids, and these enzymes therefore influence small peptide availability for antigen presentation. A better understanding of how neuropeptidases recognize their substrates could lead to therapeutics that modulate the activity of these important enzymes. Alternatively, re-engineering these enzymes to selectively hydrolyze undesirable peptides could make them attractive as therapeutics themselves. A key question in understanding the activity of these enzymes …

The Role Of Syntaxin And Tomosyn In Platelet Secretion, Shaojing Ye

Theses and Dissertations--Molecular and Cellular Biochemistry

Platelet secretion is important for hemostasis and thrombosis. The components released are also involved in atherosclerosis, inflammation, angiogenesis, and tumor growth. Though the exact mechanism(s) of platelet secretion is still elusive, accumulating evidence demonstrates that SNAREs (Soluble N-ethylmaleimide Sensitive Factor Associated Receptor) and their regulatory partners are critical for platelet exocytosis. Formation of a trans-bilayer complex composed of one v-SNARE (i.e. VAMPs) and two t-SNAREs (i.e. syntaxin and SNAP-25-type) is minimally required for membrane fusion. Regulatory proteins control the rate and specificity of the complex assembly. VAMP-8 and SNAP-23 (a SNAP-25-type t-SNARE) are clearly important; however, the …

Enantioselective Demethylation: The Key To The Nornicotine Enantiomeric Composition In Tobacco Leaf, Bin Cai

Theses and Dissertations--Plant and Soil Sciences

Nicotine and nornicotine are the two main alkaloids that accumulate in Nicotiana tabacum L. (tobacco), and nornicotine is the N-demethylation metabolite of nicotine. Nicotine is synthesized in the root, and probably primarily in the root tip. Both nicotine and nornicotine exist as two isomers that differ from each other by the orientation of H atom at the C-2' position on the pyrrolidine ring. (S)-nicotine is the dominant form in tobacco leaf and the enantiomer fraction of nicotine (EF_nic), the fraction of (R)-enantiomer over the total nicotine, is approximately 0.002. Despite considerable efforts to elucidate nicotine and nornicotine …

Investigations Into Modulation Of Brain Oxidative Stress By Various Interventions, Jessica Lynn Harris

Theses and Dissertations--Chemistry

In this thesis study we examined glycogen synthase kinase-3β (GSK-3β) and its effects over Nrf2 and Pin 1 as it relates to Alzheimer’s disease (AD). AD is a neurodegenerative disease characterized by a prolonged high oxidative environment. Transcription factor Nrf2 is vital in the brain’s defense against oxidative insults through its up-regulation of over 100 antioxidants. Depletion of the brain’s antioxidant defense system results in intolerance to an oxidative environment, contributing to the progression of AD. The regulatory Pin 1 protein promotes cellular homeostasis, and when down-regulated results in increased deposits of neurofibrillary tangles (NFTs) and amyloid-β (Aβ) plaques, the …

Bifunctional Bisphosphonates For Delivering Biomolecules To Bone, Jivan N. Yewle

Theses and Dissertations--Chemistry

Active targeting with controlled delivery of therapeutic agents to bone is an ideal approach for treatment of several bone diseases. Since bisphosphonates (BPs) are known to have high affinity to bone mineral and are being widely used in treatment of osteoporosis, they are well-suited for drug targeting to bone. For this purpose, bifunctional hydrazine-bisphosphonates (HBPs) with spacers of various lengths and lipophilicity were synthesized and studied. Crystal growth inhibition assays demonstrated that the HBPs with shorter spacers bound more strongly to bone mineral, hydroxyapatite (HA), than did alendronate. HBPs were also demonstrated to be non-toxic to MC3T3-E1 pre-osteoblasts. The targeted …

Design, Synthesis, And Anticancer Activity Of Ruthenium Complexes, Brock S. Howerton

Theses and Dissertations--Chemistry

Ruthenium complexes show promise as light activated photodynamic therapy (PDT) prodrugs. Strained octahedral complexes were synthesized that produce a cytotoxic species upon light activation. pUC19 DNA damage in vitro experiments were carried out to determine the type of damage observed. In vivo cell experiments were carried out on the non-small lung cancer A549 cell line to determine the phototherapeutic window of the synthesized complexes. One mechanism of drug resistance via elevated levels of glutathione was addressed through in vitro binding studies carried out with UV-Vis spectroscopy and in vivo glutathione titrations in the A549 cell line. Several complexes were shown …

Juvenile Hormone Regulates Vitellogenin Gene Expression Through Insulin-Like Peptide Signaling Pathway In The Red Flour Beetle, Tribolium Castaneum, Zhentao Sheng, Jingjing Xu, Hua Bai, Fang Zhu, Subba R. Palli

Entomology Faculty Publications

Our recent studies identified juvenile hormone (JH) and nutrition as the two key signals that regulate vitellogenin (Vg) gene expression in the red flour beetle, Tribolium castaneum. Juvenile hormone regulation of Vg synthesis has been known for a long time in several insects, but the mechanism of JH action is not known. Experiments were conducted to determine the mechanism of action of these two signals in regulation of Vg gene expression. Injection of bovine insulin or FOXO double-stranded RNA into the previtellogenic, starved, or JH-deficient female adults increased Vg mRNA and protein levels, thereby implicating the pivotal role for …

Investigating A Conformational Change In The Enzyme Neurolysin, Fei Xiong

Kaleidoscope

No abstract provided.

Investigating Calmodulin-Long Qt Syndrome Restorative Interactions Through Combinatorial Approaches, Michael Bricken

Kaleidoscope

No abstract provided.

Rna Oxidation Adducts 8-Ohg And 8-Oha Change With Aβ42 Levels In Late-Stage Alzheimer's Disease, Adam M. Weidner, Melissa A. Bradley, Tina L. Beckett, Dana M. Niedowicz, Amy L.S. Dowling, Sergey V. Matveev, Harry Levine, Mark A. Lovell, M. Paul Murphy

Sanders-Brown Center on Aging Faculty Publications

While research supports amyloid-β (Aβ) as the etiologic agent of Alzheimer's disease (AD), the mechanism of action remains unclear. Evidence indicates that adducts of RNA caused by oxidation also represent an early phenomenon in AD. It is currently unknown what type of influence these two observations have on each other, if any. We quantified five RNA adducts by gas chromatography/mass spectroscopy across five brain regions from AD cases and age-matched controls. We then used a reductive directed analysis to compare the RNA adducts to common indices of AD neuropathology and various pools of Aβ. Using data from four disease-affected brain …

Lysophosphatidic Acid Production And Signaling In Platelets, Zachary Bennett Fulkerson

Theses and Dissertations--Physiology

Lysophosphatidic acid (LPA) belongs to a class of extracellular lipid signaling molecules. In the vasculature, LPA may regulate platelet activation and modulate endothelial and smooth muscle cell function. LPA has therefore been proposed as a mediator of cardiovascular disease.

The bulk of circulating LPA is produced from plasma lysophosphatidylcholine (LPC) by autotaxin (ATX), a secreted lysophospholipase D (lysoPLD). Early studies suggest that some of the production of circulating LPA is platelet-dependent. ATX possesses an N-terminal somatomedin B-like domain suggesting the hypothesis that ATX interacts with platelet integrins which may localize ATX to substrate in the membrane and/or alter the catalytic …

Fabrication And Characterization Of Mesoscale Protein Patterns Using Atomic Force Microscopy (Afm), Pei Gao

University of Kentucky Doctoral Dissertations

A versatile AFM local oxidation lithography was developed for fabricating clean protein patterns ranging from nanometer to sub-millimeter scale on octadecyltrichlorosilane (OTS) layer of Si (100) wafer. This protein patterning method can generate bio-active protein pattern with a clean background without the need of the anti-fouling the surface or repetitive rinsing.

As a model system, lysozyme protein patterns were investigated through their binding reactions with antibodies and aptamers by AFM. Polyclonal anti-lysozyme antibodies and anti-lysozyme aptamer are found to preferentially bind to the lysozyme molecules on the edge of a protein pattern before their binding to the interior ones. It …

Loss Of Bloom Syndrome Protein Causes Destabilization Of Genomic Architecture And Is Complemented By Ectopic Expression Of Escherichia Coli Recg In Human Cells, Michael Wayne Killen

University of Kentucky Doctoral Dissertations

Genomic instability driven by non-allelic homologous recombination (NAHR) provides a realistic mechanism that could account for the numerous chromosomal abnormalities that are hallmarks of cancer. We recently demonstrated that this type of instability could be assayed by analyzing the copy number variation of the human ribosomal RNA gene clusters (rDNA). Further, we found that gene cluster instability (GCI) was present in greater than 50% of the human cancer samples that were tested. Here, data is presented that confirms this phenomenon in the human GAGE gene cluster of those cancer patients. This adds credence to the hypothesis that NAHR could be …

Computer Simulation Of A Hollow-Fiber Bioreactor: Heparan Regulated Growth Factors-Receptors Binding And Dissociation Analysis, Changjiang Zhang

University of Kentucky Doctoral Dissertations

This thesis demonstrates the use of numerical simulation in predicting the behavior of proteins in a flow environment.

A novel convection-diffusion-reaction computational model is first introduced to simulate fibroblast growth factor (FGF-2) binding to its receptor (FGFR) on cell surfaces and regulated by heparan sulfate proteoglycan (HSPG) under flow in a bioreactor. The model includes three parts: (1) the flow of medium using incompressible Navier-Stokes equations; (2) the mass transport of FGF-2 using convection-diffusion equations; and (3) the cell surface binding using chemical kinetics. The model consists of a set of coupled nonlinear partial differential equations (PDEs) for flow and …

15N Solid-State Nmr Detection Of Flavin Perturbation By H-Bonding In Models And Enzyme Active Sites, Dongtao Cui

University of Kentucky Doctoral Dissertations

Massey and Hemmerich proposed that the different reactivities displayed by different flavoenzymes could be achieved as a result of dominance of different flavin ring resonance structures in different binding sites. Thus, the FMN cofactor would engage in different reactions when it had different electronic structures. To test this proposal and understand how different protein sites could produce different flavin electronic structures, we are developing solid-state NMR as a means of characterizing the electronic state of the flavin ring, via the ¹⁵N chemical shift tensors of the ring N atoms. These provide information on the frontier orbitals. We propose that …