Biochemistry, Biophysics, and Structural Biology Commons^™

Preliminary Joint X-Ray And Neutron Protein Crystallographic Studies Of Ecdhfr Complexed With Folate And Nadp+, Qun Wan, Audrey Y. Kovalevsky, Mark A. Wilson, Brad C. Bennett, Paul Langan, Chris Dealwis

Department of Biochemistry: Faculty Publications

A crystal of Escherichia coli dihydrofolate reductase (ecDHFR) complexed with folate and NADP⁺ of 4 x 1.3 x 0.7 mm (3.6 mm³) in size was obtained by sequential application of microseeding and macroseeding. A neutron diffraction data set was collected to 2.0 A resolution using the IMAGINE diffractometer at the High Flux Isotope Reactor within Oak Ridge National Laboratory. A 1.6 A resolution X-ray data set was also collected from a smaller crystal at room temperature. The neutron and X-ray data were used together for joint refinement of the ecDHFR–folate–NADP⁺ ternary-complex structure in order to examine …

Molecular Mechanism Of Human Mismatch Repair Initiation, Sanghee Lee

Theses and Dissertations--Nutritional Sciences

DNA mismatch repair (MMR) is a highly conserved pathway that maintains genomic stability primarily by correcting mismatches generated during DNA replication. MMR deficiency leads to microsatellite instability (MSI), which is a hallmark of HNPCC (Hereditary Nonpolyposis Colorectal Cancer). Human mismatch repair is initiated by MutSα, a heterodimer of MSH2 and MSH6 subunits. Mismatch binding by MutSα triggers a series of downstream MMR events including interacting and communicating with other MMR proteins. The ATPase domain of MutSα is situated in the C-termini of its both subunits, and ATP binding is required for dissociation of MutSα from a mismatch. In eukaryotic cells, …

The Role Of Angiotensinogen In Atherosclerosis And Obesity, Congqing Wu

Theses and Dissertations--Nutritional Sciences

Angiotensinogen is the only known precursor in the renin-angiotensin system, a hormonal system best known as an essential regulator of blood pressure and fluid homeostasis. Angiotensinogen is sequentially cleaved by renin and angiotensin- converting enzyme to generate angiotensin II. As the major effector peptide, angiotensin II mainly function through angiotensin type 1 receptor.

Angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and more recently renin inhibitors are widely known as the 3 classic renin-angiotensin system inhibitory drugs against hypertension and atherosclerosis. Here, we developed an array of regents to explore the effects of angiotensinogen inhibition. First, we demonstrated that genetic deficiency of …

Optimization Of A Chemical Genetic Screen To Identify Druggable Targets In U138 Cells Treated With Laromustine, Kathryn Coe

Honors Theses

Laromustine is an experimental sulfonylhydrazine prodrug used in late-stage clinical studies against acute myeloid leukemia (AML) and glioblastoma multiforme (GBM). Despite initial promise for both indications, clinical trials for GBM have not been as successful as those for AML. To investigate methods for improving the effectiveness of laromustine in GBM and to learn more about the mechanism of action of laromustine, a chemical genetic screen will be conducted to identify agents that sensitize GBM cells to the anti-proliferative effects of laromustine. The library, which will include approximately 450 FDA-approved drugs, will be screened using a newly optimized high throughput assay …

An Unexpected Particle Oscillation For Electrophoresis In Viscoelastic Fluids Through A Microchannel Constriction, Xinyu Lu, Saurin Patel, Meng Zhang, Sang Woo Joo, Shizhi Qian, Amod Ogale, Xiangchun Xuan

Mechanical & Aerospace Engineering Faculty Publications

Electrophoresis plays an important role in many applications, which, however, has so far been extensively studied in Newtonian fluids only. This work presents the first experimental investigation of particle electrophoresis in viscoelastic polyethylene oxide (PEO) solutions through a microchannel constriction under pure DC electric fields. An oscillatory particle motion is observed in the constriction region, which is distinctly different from the particle behavior in a polymer-free Newtonian fluid. This stream-wise particle oscillation continues until a sufficient number of particles form a chain to pass through the constriction completely. It is speculated that such an unexpected particle oscillating phenomenon is a …

Ycf1-Mediated Cadmium Resistance In Yeast Is Dependent On Copper Metabolism And Antioxidant Enzymes, Wenzhong Wei, Nathan Smith, Xiaobin Wu, Heejeong Kim, Javier Seravalli, Oleh Khalimonchuk, Jaekwon Lee

Department of Biochemistry: Faculty Publications

Aims: Acquisition and detoxification of metal ions are vital biological processes. Given the requirement of metallochaperones in cellular copper distribution and metallation of cuproproteins, this study investigates whether the metallochaperones also deliver metal ions for transporters functioning in metal detoxification. Results: Resistance to excess cadmium and copper of the yeast Saccharomyces cerevisiae, which is conferred by PCA1 and CaCRP1 metal efflux P-type ATPases, respectively, does not rely on known metallochaperones, Atx1p, Ccs1p, and Cox17p. Copper deficiency induced by the expression of CaCRP1 encoding a copper exporter occurs in the absence of Atx1p. Intriguingly, CCS1 encoding the copper chaperone for superoxide …

The Impact Of Down Syndrome And Folate Depletion On Genomic Stabilizing Pathways Of Lymphoblastoid Cells, Khadijah Ibrahim Alnabbat

Wayne State University Theses

Understanding the role of nutrition plays in Down syndrome (DS) could help in the development and implementation of strategies that help overcome the negative consequences phenotypes of Down syndrome. Conserving genome integrity is crucial for cells to survive, and thus understanding how genetic defects induce damage to genomic DNA and impair subsequent repair of this damage is important. Evidence accumulated points to increased DNA damage and mutation accumulation associated with a decline in DNA repair capacity, Base Excision Repair (BER) in particular. Thus, the successful clinical management of DS resides in understanding the metabolic imbalance provoked by overexpression of genes …

Effects Of Statin Drugs And Tocotrienol Rich Fraction Supplementation In Chronic Hemodialysis Patients And Metabolomic Profile, Eno Latifi

Wayne State University Theses

Chronic kidney disease (CKD) is known as a heterogeneous disorder which currently is on the rise and lately has been classified as a public health issues in the United State and worldwide. CKD is an irreversible and progressive disease which can lead to kidney failure, and this is depicted by the advanced stage of the disorder when it reaches the point, that is classified as end stage of renal disease (ESRD) (Stage 5 of CKD) (eGRF <15 mL/min/ 1.73 m2 working capacity), where both organs are in a total or permanent kidney failure. End-Stage renal disease patients, on hemodialysis have been associated to experience an accelerated form of atherosclerosis, which is induced by inflammation, impairment of antioxidant system and elevated oxidative stress. Since the problem effecting ESRD patients is multifactorial, the objective of this investigation is to explore and look at the effects of supplementing with vitamin E-tocotrienol rich fraction (TRF), a micronutrient which has anti-inflammatory, antioxidant, and lipid lower capabilities into tackling these comorbid conditions experienced by this population. Therefore the aims of this investigation will be to explore changes in lipid profiles, inflammatory markers, and oxidative status, as well as look at any changes in metabolomic profiles. It was hypothesized that by supplementing with TRF a vitamin E, for 16 weeks in ESRD patients undergoing hemodialysis, it may help reverse and/or improve, oxidative status, inflammatory markers, increase antioxidants status and improve lipid profiles.

The study was double-blinded, randomized, parallel, placebo-controlled design trial, of 81 adult patients undergoing chronic hemodialysis at Great Lake Dialysis Clinic, Detroit MI, where …

Design And Study Of The Efflux Function Of The Egfp Fused Mexab-Oprm Membrane Transporter In Pseudomonas Aeruginosa Using Spectroscopy, Feng Ding, Kerry J. Lee, Ardeschir Vahedi-Faridi, Hiroshi Yoneyama, Christopher J. Osgood, Xiao-Hong Nancy Xu

Biological Sciences Faculty Publications

Multidrug membrane transporters (efflux pumps) can selectively extrude a variety of structurally and functionally diverse substrates (e.g., chemotoxics, antibiotics), leading to multidrug resistance (MDR) and ineffective treatment of a wide variety of diseases. In this study, we have designed and constructed a fusion gene (egfp-mexB) of N-terminal mexB with C-terminal egfp, inserted it into a plasmid vector (pMMB67EH), and successfully expressed it in the Δ MexB (MexB deletion) strain of Pseudomonas aeruginosato create a new strain that expresses MexA-(EGFP-MexB)-OprM. We characterized the fusion gene using gel electrophoresis and DNA sequencing, and determined its expression in live …

Monitoring Ligand-Induced Nucleic Acid Conformational Changes Using Ion Mobility Spectrometry-Mass Spectrometry, Bill Kenneth Redick

Legacy Theses & Dissertations (2009 - 2024)

Three-dimensional structures of biopolymers frequently dictate the biological role those molecules play. As such, investigation into structure of nucleic acids can provide important information pertaining to how those nucleic acids work. Many nucleic acid species, especially single-stranded RNA, fold into unique structures that allow them to function properly. Metals, and other cationic species, are often bound to the nucleic acid to make folding into the proper structure more favorable by neutralizing the negative charge on the nucleic acid imparted by the phosphate group. This investigation explores tertiary structure of nucleic acids that have been folded in the presence of ligands …

Ph Regulation And The Assessment Of Renal Injury Biomarkers In A Warm Perfusion Renal Allograft Preservation System, Aaron Meyer

Legacy Theses & Dissertations (2009 - 2024)

A shortage of viable kidneys available for transplantation exists today, as the number of individuals waiting for a kidney transplant continues to grow while the number of kidneys available each year for transplantation has remained stagnant. The development of technology that will allow for transplantation of organs that currently may be considered too damaged for transplant will provide access to a large number of donors who have experienced traumatic injury deaths resulting in loss of cardiac function. These potential donors account for the majority of all traumatic injury deaths in intensive care units, however their organs have experienced a degree …

Mechanisms Of Dna Synthesis And Fidelity By Y-Family Translesion And C-Family Replicative Polymerases, Purba Mukherjee

Legacy Theses & Dissertations (2009 - 2024)

Since the discovery of the DNA polymerase by Arthur Kornberg nearly 60 years ago, there have been great advances in understanding the involvement of polymerases in replication and repair. Years of genetic, biochemical and structural studies have lead to the classification of DNA-dependent DNA polymerases into six families: A, B, C, D, X and Y. In this work, I have focussed on two different families, C and Y. Hence this work is divided into two parts. Part one discusses the studies on Y-family polymerases. All Y-family polymerases are involved in replicating past DNA lesions. The ability to tolerate unnatural nucleotides …

Modeling The Early, Aging-Related Changes In Human Recombinant Gammas-Crystallin In Vitro, Christiana Oluwaseun Salami

Legacy Theses & Dissertations (2009 - 2024)

Cataract is the leading cause of blindness in the world. Cataractous lenses scatter a significant part of the light entering the eye. Cataract can be of several types: Age-related cataract, secondary cataract, traumatic cataract, congenital cataract, and radiation cataract. Age-related cataract is the most common type of cataract. Several factors contribute to age-onset cataract; among them are the Post-Translational Modifications (PTMs) to the major lens proteins or crystallins, which constitute about 80-90% of the protein content of the lens. Predominant PTMs of the crystallins are oxidation, deamidation, phosphorylation, glycation, and disulfide crosslinking, which tend to increase as the lens ages.

Mechanisms Of Age-Related Inflammation And Cancer : The Synergistic Effect Of Oxidants And Calcium, Donald A. Mccarthy

Legacy Theses & Dissertations (2009 - 2024)

The accumulation of senescent cells during the process of aging has been implicated as causal in numerous age-related pathologies. Senescent cells adopt a secretory phenotype consisting of many factors including matrix remodeling enzymes, growth factors, cytokines, and chemokines. Their secretory nature is the primary reason that they are associated with disease, but it remains unclear why they become so inflammatory. Using primary human fibroblasts cultured to senescence, we mechanistically determined why senescent cells are such potent inducers of inflammation. Our findings indicate that the early production of the cytokine Interleukin 1-α (IL-1α) is central to this transition. We found that …

Probing Secondary And Tertiary Rna Folding Using Force And Temperature, William Stephenson

Legacy Theses & Dissertations (2009 - 2024)

RNA folding is the process whereby a single stranded RNA molecule assumes its three-dimensional functional conformation. Along with the protein folding problem, the RNA folding problem remains as one of the great unsolved problems in biophysics. Generally RNA folding occurs in a hierarchical manner whereby the sequence of an RNA (primary structure) determines which regions will form helical segments (secondary structure) before further rearrangement and base pairing of secondary structure motifs (tertiary structure). Due to the intimate connection between structure and function within molecular biology, increased familiarity with the thermodynamic and kinetic factors that govern RNA folding will permit the …

Mechanistic Studies Of C- And Y-Family Dna Polymerases, Indrajit Lahiri

Legacy Theses & Dissertations (2009 - 2024)

DISSERTATION ABSTRACT

Raman Spectroscopy Of Blood Serum And Cerebrospinal Fluid And Multivariate Data Analysis For Alzheimer's Disease Diagnostics, Elena Ryzhikova

Legacy Theses & Dissertations (2009 - 2024)

The efficient and accurate diagnosis at the early stages of dementia is a key moment for effective treatment and productive research to find a new ways to combat the disease. It is especially true for Alzheimer's disease (AD) for which there is no effective cure, but several treatments are known to allow slowing down the degenerative processes. Alzheimer's disease (AD) displays only non-specific clinical symptoms of mental decline for decades after the initiation and is very challenging to differentiate even at the later stages when it becomes very aggressive. Despite the great need, current diagnostic tests are unable to diagnose …

A Structural And Functional Comparison Of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein (Ncp7) And Other Retroviral Nucleocapsid Proteins, Christopher Richard Cavender

Legacy Theses & Dissertations (2009 - 2024)

Human immunodeficiency virus type 1 (HIV-1) is a retrovirus belonging to the lentivirus genus of the orthoretrovirinae family. HIV-1 is widely known for causing acquired immunodeficiency syndrome (AIDS) in humans and being responsible for 1.8 million deaths due to AIDS associated illnesses in 201016.

Quantification Of Free Sialic Acid In Human Plasma Through A Robust Quinoxalinone Derivatization And Lc–Ms/Ms Using Isotope-Labeled Standard Calibration, Dan Wang, Xiang Zhou, Lin Wang, Sihe Wang, Xue-Long Sun

Chemistry Faculty Publications

We report an accurate quantification of free sialic acid (SA) in human plasma using LC–MS/MS method with isotope-labeled standard calibration (ILSC) and robust derivatization. Specifically, derivatization of SA with a stable and inexpensive 3,4-diaminotoluene (DAT) provides a stable product of SA with high MS response, proving a convenient and cost-effective LC–MS/MS analysis of free SA. In addition, the use of ¹³C₃-SA as calibration standard ensured the accuracy for the measurement. This assay used ultra high performance liquid chromatography (UHPLC) for separation of native/labeled SA and IS from matrix interference, and employed mass spectrometry in multiple reaction monitoring …

Identification And Characterization Of Human Plasma Prekallikrein-Prolylcarboxypeptidase Interaction Sites, Jingjing Wang

Electronic Theses and Dissertations

Prolylcarboxypeptidase isoform1 (PRCP1, also known as angiotensinase C, PCP, PRCP, PrCP) is a widely distributed serine protease found throughout the human body. PRCP1 removes the C-terminal amino acid which is next to a proline residue of a peptide. Through this activity, PRCP1 is postulated to play distinct biological roles including the regulation of vascular homeostasis, the induction of inflammation and the adjustment of metabolism. Compelling evidence indicates that human PRCP1 activates plasma prekallikrein (PK) to kallikrein on endothelial cells. However, the mechanism of this activation is yet unknown. The formation of kallikrein leads to the generation of proinflammatory factors and …

Comparative Phenolic Content And Antioxidant Activities Of Blueberry (Vaccinium Corymbosum) Afected By In Vitro Digestion, Sonia Gharbi

Wayne State University Theses

Abstract

COMPARATIVE PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITIES OF BLUEBERRY (VACCINIUM CORYMBOSUM) AFECTED BY IN VITRO DIGESTION

By

SONIA GHARBI

May 2014

Adviser: Dr. Kevin Zhou

Major: Nutrition and Food Science

Degree: Master of Science

The biological properties of antioxidants depend on their release from the food matrix during the digestion process. Blueberry contains a wide range of phenolic compounds which are of great significance due to their antioxidant activity. In vitro digestion is a rapid and inexpensive method used to determine the availability of nutrients involved in the absorption studies with humans. Total phenolic content and antioxidant activity of Vaccinium …

In Vitro Characterization Of Serine 47 Phosphorylated Cytochrome C, Ashwathy Mary Varughese

Wayne State University Theses

Cytochrome c is a 12.4kDa ubiquitously expressed protein known to be involved in many physiological processes of the cell such as respiration and apoptosis. The goal of our lab is to increase our knowledge of the regulation of cytochrome c in these opposite activities, and our working model posits that cytochrome c is decisively regulated by phosphorylation. When phosphorylated, cytochrome c leads to an "optimal" functioning in the electron transport chain by lowering electron flux, preventing harmful high mitochondrial membrane potentials and thus ROS production under healthy conditions. However, under cellular stress cytochrome c might be dephosphorylated favoring high mitochondrial …

Biophysical Studies Of Mutant H69 Rna Constructs, Daya Nidhi Kharel

Wayne State University Theses

The importance of helix 69 (H69) of the large ribosomal subunit is emphasized by its location in the ribosome and key contacts with protein factors and tRNAs during different steps of protein synthesis. The presence of three highly conserved pseudouridine modifications in H69 has a significant impact on modulation of the ribosome conformations. Specific nucleotides of H69 may be necessary for the function of this helix-loop RNA, as some mutations are deleterious to bacterial growth. In this study, we investigated the effects of single nucleotide mutations in a 19-nucleotide H69 construct with and without pseudouridines on the RNA stability and …

Effect Of Long Term Rapamycin Treatment On Mtor Signalling Network In Colon And Liver Of C57bl/6 Mice, John Sorge

Wayne State University Theses

Many studies have investigated the effects of rapamycin on aging and cancer. However, the effects of long-term rapamycin supplementation on a cancer model have not been performed. This is the first study that investigates the effects of long-term supplementation of rapamycin in a cancer model. ACF analysis of colon tissues in mice showed no significant difference between controls and those supplemented with rapamycin. Factors such as energy balance, cellular environment, PI3K/Akt/mTOR pathway, and more have been assessed in this study. The duration of rapamycin supplementation seems to play an important role in the protection against cancer. Ultimately, this study suggests …

Genetic And Biochemical Studies Of Human Apobec3h Enzyme, Thisari Sachithra Aluthgama Guruge

Wayne State University Theses

The AID/APOBEC enzymes are DNA/RNA cytosine deaminases with important functions in innate and adaptive immunity. APOBEC3 enzymes play a crucial role in restricting the replication of exogenous retroviruses such HIV-1 and endogenous retrotransposition events. In particular, APOBEC3 enzymes have evolved in humans by gene duplication to compose seven members. APOBEC3H is highly diverse in its allelic sequence and the distribution is population-specific. It has evolved under strong positive selection over millions of years. To study the catalytic mechanism, structure, function and the underlying cause of its high diversity, a high resolution crystal or NMR structure is required are not yet …

Microfluidic Electrical Sorting Of Particles Based On Shape In A Spiral Microchannel, John Dubose, Xinyu Lu, Saurin Patel, Shizhi Qian, Sang Woo Joo

Mechanical & Aerospace Engineering Faculty Publications

Shape is an intrinsic marker of cell cycle, an important factor for identifying a bioparticle, and also a useful indicator of cell state for disease diagnostics. Therefore, shape can be a specific marker in label-free particle and cell separation for various chemical and biological applications. We demonstrate in this work a continuous-flow electrical sorting of spherical and peanut-shaped particles of similar volumes in an asymmetric double-spiral microchannel. It exploits curvature-induced dielectrophoresis to focus particles to a tight stream in the first spiral without any sheath flow and subsequently displace them to shape-dependent flow paths in the second spiral without any …

Biochemical Characterization Of Four Distinct Proteins, Gustavo A. Avila

Open Access Theses & Dissertations

Proteins are ubiquitous in all living organisms, executing the majority of cellular functions in distinct ways. Understanding a protein's role necessitates investigating its structure and function, which are closely related. My research couples these two aspects by delving into the biochemical and structural characterization of proteins in four distinct systems, all playing central roles in numerous significant disease progressions. These four original research endeavors were all targeted for structural studies with a unifying relationship to establish our new structural biochemistry lab. These four systems are: (1) Gam1, an early adenovirus protein globally inhibiting host SUMOylation; (2) Anthrax toxin complexed with …

Investigating Structure And Protein-Protein Interactions Of Key Post-Type Ii Pks Tailoring Enzymes, Theresa E. Downey

Theses and Dissertations--Pharmacy

Type II polyketide synthase (PKS) produced natural products have proven to be an excellent source of pharmacologically relevant molecules due to their rich biological activities and chemical scaffolds. Type II-PKS manufactured polyketides share similar polycyclic aromatic backbones leaving their diversity to stem from various chemical additions and alterations facilitated by post-PKS tailoring enzymes. Evidence suggests that post-PKS tailoring enzymes form complexes in order to facilitate the highly orchestrated process of biosynthesis. Thus, protein-protein interactions between these enzymes must play crucial roles in their structures and functions. Despite the importance of these interactions little has been done to study them. In …

Simultaneous Bactericidal And Osteogenic Effect Of Nanoparticulate Calcium Phosphate Powders Loaded With Clindamycin On Osteoblasts Infected With Staphylococcus Aureus, Vuk Uskoković, Tejal A. Dasai

Pharmacy Faculty Articles and Research

S aureus internalized by bone cells and shielded from the immune system provides a reservoir of bacteria in recurring osteomyelitis. Its targeting by the antibiotic therapy may thus be more relevant for treating chronic bone infection than eliminating only the pathogens colonizing the bone matrix. Assessed was the combined osteogenic and antibacterial effect of clindamycinloaded calcium phosphate nanoparticles of different monophasic compositions on co-cultures comprising osteoblasts infected with S aureus. Antibiotic-carrying particles were internalized by osteoblasts and minimized the concentration of intracellular bacteria. In vitro treatments of the infected cells, however, could not prevent cell necrosis due to the …

Β-Lactam Estrogen Receptor Antagonists And A Dual-Targeting Estrogen Receptor/Tubulin Ligand, Niamh O'Boyle, Jade K. Pollock, Miriam Carr, Andrew Js Knox, Seema M. Nathwani, Shu Wang, Laura Caboni, Daniela M. Zisterer, Mary Meegan

Articles

Twelve novel β-lactams were synthesised and their antiproliferative effects and binding affinity for the predominant isoforms of the estrogen receptor (ER), ERα and ERβ, were determined. β-Lactams 23 and 26 had the strongest binding affinities for ERα (IC₅₀ values: 40 and 8 nM respectively) and ERβ (IC₅₀ values: 19 and 15 nM). β-Lactam 26 was the most potent in antiproliferative assays using MCF-7 breast cancer cells, and further biochemical analysis showed that it caused accumulation of cells in G₂/M phase (mitotic blockade) and depolymerisation of tubulin in MCF-7 cells. Compound 26 also induced apoptosis and downregulation …