Biochemistry, Biophysics, and Structural Biology Commons^™

Analysis Of Biomolecular Interactions Using Affinity Microcolumns: A Review, Xiwei Zheng, Zhao Li, Sandya Beeram, Maria Podariu, Ryan Matsuda, Erika L. Pfaunmiller, Christopher J. White Ii, Natasha Carter, David S. Hage

David Hage Publications

Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and …

Glycosyl Rotation And Distortion By Key Residues In Endocellulase Cel6a From Theromobifida Fusca, Tao Lu, Zuoming Zhang, Chi Zhang

School of Biological Sciences: Faculty Publications

Endocellulases are one kind of the important biodegrading cellulose enzymes. Experimental results show that a rotated and distorted preactivated structure exists before the substrate entering the transition state. The molecular dynamic simulation of endocellulase Cel6A models revealed a correlation between the rotation and distortion of pyranoside ring in −1 glycosyl unit of the substrate. The two key residues, Tyr73 and Ser189, in Cal6A cooperate to rotate and distort the pyranoside ring in the cellulose hydrolysis.

Includes supplementary material.

Further Characterization Of A Secreted Lipase From The Human Pathogen Leishmania Donovani By Determining The Effect Of Various Metal Ions On Its Enzymatic Activity., Lana Hoertz

Pell Scholars and Senior Theses

Leishmania donovani, a protozoan parasite, is the causative agent of the often fatal disease visceral leishmaniasis. The current treatments available are minimal and toxic to the patient. It has been shown that these organisms exhibit lipolytic activity during their growth in vitro. Lipases are enzymes that are known to aid in the development and virulence of several pathogenic organisms such as Candida albicans and Staphylococcus warneri. Little information is known, however, about the role of lipases in Leishmania species. We hypothesize that lipase may play a part in Leishmania's ability to survive within the human host as well as its …

Culturable Aerobic And Facultative Anaerobic Intestinal Bacterial Flora Of Black Cobra (Naja Naja Karachiensis) In Southern Pakistan, Junaid Iqbal, Mehwish Sagheer, Nazneen Tabassum, Ruqaiyyah Siddiqui, Naveed Ahmed Khan

Department of Biological & Biomedical Sciences

Using morphological analysis and biochemical testing, here for the first time, we determined the culturable gut bacterial flora (aerobes and facultative anaerobes) in the venomous Black Cobra (Naja naja karachiensis) from South Asia. The findings revealed that these snakes inhabit potentially pathogenic bacteria including Serratia marcescens, Pseudomonas aeruginosa, Shewanella putrefaciens, Aeromonas hydrophila, Salmonella sp., Moraxella sp., Bacillus sp., Ochrobactrum anthropi, and Providencia rettgeri. These findings are of concern, as injury from snake bite can result in wound infections and tissue necrosis leading to sepsis/necrotizing fasciitis and/or expose consumers of snake meat/medicine in the community to infections.

Synthesis And Characterization Of Pt(Ii) Complexes For Anticancer Therapy, Mihaela A. Ciulei, Pradip K. Bhowmik

McNair Poster Presentations

The first platinum-based drug was discovered and approved by Food and Drug Administration (FDA) in 1978 is cis-diamminedichloroplatinum (II) (cisplatin or CDDP). Cisplatin is used for about 50% of the chemotherapeutic cancer treatments along with its two analogues carboplatin and oxaliplatin. So far these drugs have been used extensively as treatment for ovarian, bladder, head and neck, and lung cancers. Although cisplatin has been used so often, it has toxic side effects and drug resistance.1-4 Due to these limitations other compounds have been synthesized. Specifically, our lab in conjunction with a biochemistry lab has recently published one article …

A Loose Domain Swapping Organization Confers A Remarkable Stability To The Dimeric Structure Of The Arginine Binding Protein From Thermotoga Maritima, Alessia Ruggiero, Jonathan D. Dattelbaum, Maria Staiano, Rita Berisio, Sabato D'Auria, Luigi Vitagliano

Chemistry Faculty Publications

The arginine binding protein from Thermatoga maritima (TmArgBP), a substrate binding protein (SBP) involved in the ABC system of solute transport, presents a number of remarkable properties. These include an extraordinary stability to temperature and chemical denaturants and the tendency to form multimeric structures, an uncommon feature among SBPs involved in solute transport. Here we report a biophysical and structural characterization of the TmArgBP dimer. Our data indicate that the dimer of the protein is endowed with a remarkable stability since its full dissociation requires high temperature as well as SDS and urea at high concentrations. In order to elucidate …

Functions Of Mitoneet: What The Protein-Protein Interactions Reveal, Fran Hirschfelder

Masters Theses

MitoNEET is a [2Fe-2S] cluster protein found on the outer mitochondrial membrane. First discovered as a binding site for pioglitazone, an anti-diabetic drug, it was the first protein described containing a CDGSH iron-sulfur domain (CISD). However, the function(s) of mitoNEET in both normal cellular physiology and the physiology in type-2 diabetes remains unclear. The proposed functions of mitoNEET, include cluster transfer, electron transport and oxidative respiration. This thesis describes the use of proteomic pull-down techniques to identify possible protein binding partners of mitoNEET from both mouse and human liver cell lysates. In order to accomplish this, the expression and purification …

Role Of Cardiac Catecholamines In Embryos And Adults Under Stress, Candice Baker

Electronic Theses and Dissertations

Cardiovascular disease is responsible for the loss of one life every 38 seconds and accounts for 26.6 percent of all infants that die of congenital birth defects. Adrenergic hormones are critically important regulators of cardiovascular physiology in embryos and adults. They are key mediators of stress responses and have profound stimulatory effects on cardiovascular function, and dysregulation of adrenergic function has been associated with many adverse cardiac conditions, including congenital malformations, arrhythmias, ischemic heart disease, heart failure, and sudden cardiac death. Despite intensive study, the specific roles these hormones play in the developing heart is not well-understood. Further, there is …

In Vitro Characterization Of Unmodified And Pyroglutamylated Alzheimer's Amyloid Beta Peptide, Jason Matos

Electronic Theses and Dissertations

Plaques of amyloid β peptide (Aβ) are a hallmark trait of Alzheimer’s disease (AD). However, the precise role of Aβ aggregates is not well understood. Recent studies have identified that naturally occurring N-terminal truncation and pyroglutamylation of Aβ significantly increases its neurotoxicity by an unknown mechanism. Content of pyroglutamylated Aβ (pE-Aβ) in AD brains has been shown to reach up to 50% of total Aβ. Modified pE-Aβ co-aggregates with Aβ by a seeding mechanism and forms structurally distinct and highly toxic oligomers. We studied structural transitions of the full-length Aβ1-42, its pyroglutamylated form AβpE3-42, their 9:1 (Aβ1-42/AβpE3-42) and 1:1 molar …

Establishment Of Methods For Isolation Of Pnmt+ Cardiac Progenitor Cells, Namita Varudkar

Electronic Theses and Dissertations

Cardiovascular disease is the leading cause of death in the United States. Millions of patients suffer each year from endothelial dysfunction and/or debilitating myocardial damage resulting in decreased quality of life and increased risk of death or disablement. Current pharmacological approaches are only partly effective at treating cardiovascular disease, and hence, better strategies are needed to provide significant improvements in treatment options. Cardiac stem/progenitor cells have the potential to regenerate myocardial tissue and repair damaged heart muscle. There are many different types of cardiac progenitor cells, and each may have certain unique properties and characteristics that would likely be useful …

Genetic And Biochemical Characterization Of The Roles Of Two Putative Purine Transporters In The Infectious Cycle Of Borrelia Burgdorferi, Sunny Jain

Electronic Theses and Dissertations

Lyme disease, the most common tick borne disease in United States, is caused by the bacterial pathogen Borrelia burgdorferi. In nature, B. burgdorferi exists in an enzootic infectious cycle between an arthropod vector and mammalian hosts. Identification and characterization of the genes essential for B. burgdorferi survival throughout its infectious cycle is an important step toward understanding the molecular mechanisms involved in B. burgdorferi pathogenesis. B. burgdorferi contains a small genome, which lacks the genes encoding for the enzymes required for de novo synthesis of amino acids, fatty acids and nucleic acid precursors. Therefore, the spirochete is dependent upon the …

Bipolar Nanosecond Electric Pulses Are Less Efficient At Electropermeabilization And Killing Cells Than Monopolar Pulses, Bennett L. Ibey, Olga N. Pakhomova, Caleb C. Roth, Shu Xiao, Karl Schoenbach, Andrei G. Pakhomov

Bioelectrics Publications

Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, propidium iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium …

Validation Of A Novel Hypothesis Of Generating Foam Cells By Its Use To Study Reverse Cholesterol Transport, Bhaswati Sengupta

Electronic Theses and Dissertations

Generation of foam cells, an essential step for reverse cholesterol transport (RCT) studies, uses the technique of receptor dependent macrophage loading with radiolabeled acetylated Low Density Lipoprotein (Ac-LDL). In this study, we used the ability of a biologically relevant detergent molecule, Lysophosphatidylcholine (Lyso PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabelled cholesterol / Lyso PtdCho mixed micelles were prepared and incubated with RAW 264.7 or mouse peritoneal macrophages. Results showed that such micelles were quite stable at 4°C and retained the solubilized cholesterol during one month storage. Macrophages incubated …

Cerium Oxide Nanoparticles: Their Phosphatase Activity And Its Control, Atul Dhall

Electronic Theses and Dissertations

Cerium oxide nanoparticles are established scavengers of reactive oxygen and nitrogen species. They have many potential biomedical applications that depend on their physicochemical properties and mode of preparation. Recent studies have found these nanoparticles possess phosphatase mimetic activity. Studying such catalytic activities will qualify their biomedical applications and render information on their bioavailability and potential toxicity. Two oxidation states of cerium exist in these nanoparticles (3+ or 4+). It is hypothesized that the oxidation state of cerium in the nanoparticles determines the amount of adsorbed water on the crystal lattices. This in turn governs their activity as phosphatases. Nanoparticles with …

A Theoretical Study Of Interaction Of Nanoparticles With Biomolecule, Chunhui Liu

Dissertations, Master's Theses and Master's Reports - Open

Many types of materials at nanoscale are currently being used in everyday life. The production and use of such products based on engineered nanomaterials have raised concerns of the possible risks and hazards associated with these nanomaterials. In order to evaluate and gain a better understanding of their effects on living organisms, we have performed first-principles quantum mechanical calculations and molecular dynamics simulations. Specifically, we will investigate the interaction of nanomaterials including semiconducting quantum dots and metallic nanoparticles with various biological molecules, such as dopamine, DNA nucleobases and lipid membranes.

Firstly, interactions of semiconducting CdSe/CdS quantum dots (QDs) with the …

Enzyme Optimization For Lignocellulose Hydrolysis Using Mechanistic Modeling, Adam S. Marlowe

Dissertations, Master's Theses and Master's Reports - Open

A novel mechanistic model for the saccharification of cellulose and hemicellulose is utilized to predict the products of hydrolysis over a range of enzyme loadings and times. The mechanistic model considers the morphology of the substrate and the kinetics of enzymes to optimize enzyme concentrations for the enzymatic hydrolysis of cellulose and hemicellulose simultaneously. Substrates are modeled based on their fraction of accessible sites, glucan content, xylan content, and degree of polymerizations. This enzyme optimization model takes into account the kinetics of six core enzymes for lignocellulose hydrolysis: endoglucanase I (EG1), cellobiohydrolase I (CBH1), cellobiohydrolase II (CBH2), and endo-xylanase (EX) …

Evaluations Of A Mechanistic Hypothesis For The Influence Of Extracellular Ions On Electroporation Due To High-Intensity, Nanosecond Pulsing, V. Sridhara, R. P. Joshi

Electrical & Computer Engineering Faculty Publications

The effect of ions present in the extracellular medium on electroporation by high-intensity, short-duration pulsing is studied through molecular dynamic simulations. Our simulation results indicate that mobile ions in the medium might play a role in creating stronger local electric fields across membranes that then reinforce and strengthen electroporation. Much faster pore formation is predicted in higher conductivity media. However, the impact of extracellular conductivity on cellular inflows, which depend on transport processes such as electrophoresis, could be different as discussed here. Our simulation results also show that interactions between cations (Na+ in this case) and the carbonyl oxygen of …

Evolution And Divergence Of The Structural And Physical Properties Of Dna Binding By Methyl-Cytosine Binding Domain Family Members 2 And 3, Jason Cramer

Theses and Dissertations

The studies presented in this dissertation, Evolution And Divergence Of The Structural And Physical Properties Of DNA Binding By Methyl-Cytosine Binding Domain Family Members 2 And 3, pertain primarily to two key epigenetic regulators involved with the biological interpretation of methylated DNA marks. We provide insights into the emergence and evolution of the MBD2 and MBD3 and how those molecular entities influence heritable changes in gene activity. We further provide details regarding the mystery surrounding MBD3 function and the MBD2-mediated capacity of primitive animals to carry out methylation-specific epigenetic mechanisms. In chapter two, we describe the DNA binding properties of …

Structural Studies Of Interferon Regulatory Factor 4: A Molecular Perspective Of Its Regulatory Mechanism, Soumya Govinda Remesh

Theses and Dissertations

Interferon (IFN) regulatory factor family member 4 (IRF4) is a transcription factor that serves specific roles in transcriptional regulation of IFN responsive genes and is indispensable in B- & T-cell differentiation. IRF4 like the other members of the family has two major domains- the N-terminal DNA binding domain (DBD) essential for its recognition and binding to the Interferon Stimulated Response Element DNA sequence and a C-terminal Interferon activation domain (IAD) thought to maintain IRF4 in an auto-inhibited inactive state and is also critical in its activation. A putative unstructured linker connects the DBD and IAD. Activation in most members of …

A Summary Of Three Antibiotic Resistant Bacteria Studies Of Plaster Creek, Yeaeun Lee, David P. Warners

Faculty and Professional Research

No abstract provided.

Nine Year Assessment Of Soil Treatment On Aguacatillo (Nectandontisii And Ocotea Austinii, Lauraceae) Seedling Success In The Restoration Of A Costa Rican Cloud Forestra Cufo, Sheila Devries, David Warners

Faculty and Professional Research

We tracked seedling growth of two species of aguacatillo (Nectandra cufodontisii (O. C. Schmidt) C. K. Allen and Ocotea austinii C. K. Allen) that were planted into an early successional cloud forest hillside in San Gerardo de Dota, Costa Rica over nine years. Seedlings were collected from underneath nearby mother trees and transplanted into one of three soil treatments: indigenous soil; a 50:50 mixture of horse manure and indigenous soil; and a 50:50 mixture of kitchen compost and indigenous soil. After seedlings were transplanted they were measured and each specific location was evaluated for light exposure. Two months later seedlings …

Single-Molecule Analysis Of Alzheimer's Β-Peptide Oligomer Disassembly At Physiological Concentration, Chen Chen

Theses and Dissertations--Chemistry

The diffusible soluble oligomeric amyloid β-peptide (Aβ) has been identified as a toxic agent in Alzheimer’s disease that can cause synaptic dysfunction and memory loss, indicating its role as potential therapeutic targets for AD treatment. Recently an oligomer-specific sandwich biotin-avidin interaction based assay identified the Aβ oligomer dissociation potency of a series of dihydroxybenzoic acid (DHBA) isomers. Because the sandwich assay is an ensemble method providing limited size information, fluorescence correlation spectroscopy (FCS) was employed to provide single molecule resolution of the disassembly mechanism.

Using FCS coupled with atomic force microscopy, we investigated the size distribution of fluorescein labeled synthetic …

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 …

Bases For Breadth - Insights Into How The Mechanism And Dynamics Of Nitroreductase Can Explain This Enzyme's Broad Substrate Repertoire, Warintra Pitsawong

Theses and Dissertations--Chemistry

Nitroreductase from Enterobacter cloacae (NR) is a member of a large family of homologues represented in all branches of the tree of life. However the physiological roles of many of these enzymes remain unknown. NR has distinguished itself on the basis the diverse sizes and chemical types of substrates it is able to reduce (Koder et al 1998). This might be an evolved characteristic suiting NR for a role in metabolism of diverse occasional toxins. While there are numerous studies of determinants of substrate specificity, we know less about mechanisms by which enzymes can be inclusive. Therefore, we present …

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 …

Bioc 435: Advanced Topics In Biochemistry—A Peer Review Of Teaching Project Benchmark Portfolio, Edward N. Harris

UNL Faculty Course Portfolios

BIOC 435 or Advanced Topics in Biochemistry is an ACE 10 (Achievement centered education)course for students in their senior year. This is a capstone course for Biochemistry majors and is the last of several biochemistry courses that they will take in their undergraduate enrollment. The course is also open to non-majors in a related science such as Biology and Biological Sciences, although these students typically do not do as well as the majors. Students who take this course should be well-prepared in the basic concepts of biochemistry with regards to nucleic acids and proteins as well as some familiarity with …

High Fat Diet Increased Serum Glutamate Dehydrogenase More Than Chronic Acetaminophen Dosing In Female Mice, Elizabeth Ruth Behmer

All Graduate Theses, Dissertations, and Other Capstone Projects

This laboratory study examined the effects of acetaminophen overdose in normal fed and high-fat fed female mice. Forty female mice were placed on normal and high-fat diets at 4 weeks old. When the mice were significantly different in weight (between 6 and 9 months old), half the mice were dosed with acetaminophen. These mice were daily given an overdose of acetaminophen for 14 days. The dose used was 300 mg/kg mouse (LD50 = 338 mg/kg). The control group was given 10 µl water/g mouse. Levels of serum glutamate dehydrogenase (GDH) were measured to indicate liver damage. GDH is released from …

Modulation Of Cholera Toxin Structure And Function By Host Proteins, Helen Burress

Electronic Theses and Dissertations

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic thermal instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the ER-to-cytosol translocation of CTA1, yet the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Potential post-translocation roles for Hsp90 …

Dimethylarginine Dimethylaminohydrolase Ii Overexpression Attenuates Lps-Mediated Lung Leak In Acute Lung Injury, Saurabh Aggarwal, Christine M. Gross, Sanjiv Kumar, Christiana Dimitropoulou, Shruti Sharma, Boris A. Gorshkov, Supriya Sridhar, Qing Lu, Natalia V. Bogatcheva, Agnieszka J. Jezierska-Drutel, Rudolf Lucas, John D. Catravas, Stephen M. Black

Bioelectrics Publications

Acute lung injury (ALI) is a severe hypoxemic respiratory insufficiency associated with lung leak, diffuse alveolar damage, inflammation, and loss of lung function. Decreased dimethylaminohydrolase (DDAH) activity and increases in asymmetric dimethylarginine (ADMA), together with exaggerated oxidative/nitrative stress, contributes to the development of ALI in mice exposed to LPS. Whether restoring DDAH function and suppressing ADMA levels can effectively ameliorate vascular hyperpermeability and lung injury in ALI is unknown, and was the focus of this study. In human lung microvascular endothelial cells, DDAH II overexpression prevented the LPS-dependent increase in ADMA, superoxide, peroxynitrite, and protein nitration. DDAH II also attenuated …