Biochemistry, Biophysics, and Structural Biology Commons^™

Hydrogen Peroxide Probes Directed To Different Cellular Compartments, Mikalai Malinouski, You Zhou, Vsevolod V. Belousov, Dolph L. Hatfield, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Background: Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells.

Principal Findings: Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed …

Targeted Deletion Of The Mouse Mitoferrin1 Gene: From Anemia To Protoporphyria, Marie-Berengere Troadec, David Warner, Jared Wallace, Kirk Thomas, Gerald J. Spangrude, John Phillips, Oleh Khalimonchuk, Barry H. Paw, Diane Mcvey Ward, Jerry Kaplan

Department of Biochemistry: Faculty Publications

Mitoferrin1 is 1 of 2 homologous mitochondrial iron transporters and is required for mitochondrial iron delivery in developing erythroid cells. We show that total deletion of Mfrn1 in embryos leads to embryonic lethality. Selective deletion of Mfrn1 in adult hematopoietic tissues leads to severe anemia because of a deficit in erythroblast formation. Deletion of Mfrn1 in hepatocytes has no phenotype or biochemical effect under normal conditions. In the presence of increased porphyrin synthesis, however, deletion of Mfrn1 in hepatocytes results in a decreased ability to convert protoporphyrin IX into heme, leading to protoporphyria, cholestasis, and bridging cirrhosis. Our results show …

The Role Of Mdm2 In Estrogen-Mediated Breast Cancer Cell Proliferation, Angelika Brekman

Dissertations, Theses, and Capstone Projects

Estrogen signaling is important in breast cancer development and progression. Mdm2, a negative regulator of the p53 tumor suppressor, is often over-expressed in estrogen receptor positive breast cancers. To study the role of Mdm2 in the estrogen-mediated breast cancer cell proliferation, we examined the effect of estrogen on the p53-Mdm2 pathway in estrogen receptor positive and p53 wild-type MCF-7 breast cancer cells. Estrogen-mediated increase in cell proliferation correlated with increased Mdm2, but no concomitant decrease in the p53 protein level. Blocking Mdm2 expression with inducible shRNA inhibited estrogen-mediated cell proliferation and colony formation in soft agar. Mdm2 knockdown in the …

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 …

Infection With Chlamydia Pneumoniae In Neuronal Cells Alters The Expression Of Genes Involved In Apoptosis And Autophagy Pathways, Annette K. Slutter

PCOM Biomedical Studies Student Scholarship

Dysfunctions in cellular mechanisms such as apoptosis and autophagy have been implicated in the neurodegeneration associated with Alzheimer’s disease (AD). Autophagy in AD pathogenesis has been linked to the endosomal-lysosomal system, which has been shown to play a role in amyloid processing. Studies have suggested that apoptosis may contribute to the neuronal cell loss observed in AD; however, there is no evidence of the apoptotic process leading to terminal completion. Aβ1-42 has been shown to induce apoptosis in neurons and may be an initiating factor in AD. Our previous studies demonstrated that neurons infected with C. pneumoniae are resistant to …

Cardiac Calsequestrin Phosphorylation And Trafficking In The Mammalian Cardiomyocyte, Timothy Mcfarland

Wayne State University Dissertations

Cardiac CSQ (CSQ2) is a multifaceted protein, capable of binding significant quantities of Ca2+ and altering ryanodine receptor activity at the junctional sarcoplasmic reticulum (SR). Little is known about the trafficking of CSQ2 from its unknown site of biosynthesis, which appears to be of importance as its structure changes in a trafficking-dependent manner in various types of heart failure. Through the use of multiple antibodies specific to classic rough ER markers, and with the creation of CSQ-DsRed tetramer fusion protein, we were able to establish a juxtanuclear localization of rough ER in cardiomyocytes. Using fluorescence confocal microscopy, the translocon complex …

Crystallographic, Molecular Dynamics, And Enzymatic Studies Of Multi-Drug Resistant Hiv-1 Protease And Implications For Structure Based Drug Design (Project 1); Crystallographic Studies Of Human Myelin Protein Zero (Project 2), Zhigang Liu

Wayne State University Dissertations

Under drug selection pressure, emerging mutations render HIV-1 protease drug resistance, leading to the therapy failure in anti-HIV treatment.Tthe multidrug-resistant 769 (MDR) HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) is selected for the present study to understand drug resistance issue.

Ten additional mutations are introduced to MDR769 HIV-1 protease to study the structural influences brought by these mutations. We get crystal structures of four variants (I10V, A82F, A82S and A82T) of MDR769 HIV-1 protease. All these mutations fail to further open the flaps and expand the active site cavity of MDR769 …

Amphiregulin (Areg) And Epidermal Growth Factor (Egf): Disparate In Egfr Signaling And Trafficking, Andrea Jacqueline Baillo

Wayne State University Dissertations

We have previously shown that SUM-149 human breast cancer cells require an AREG/EGFR autocrine loop for cell proliferation. We also demonstrated that AREG can increase EGFR stability and promote EGFR localization to the plasma membrane. In the presented dissertation we successfully knocked-down AREG expression in SUM-149 cells by lenti-viral infection of AREG shRNA. In the absence of AREG expression, SUM-149 cell growth was slowed, but not completely inhibited. Furthermore, cells infected with AREG shRNA constructs showed an increase in EGFR protein expression by western blot. Immunofluorescence and confocal microscopy showed that following AREG knock-down, EGFR continued to localize to the …

Frazzled And Abelson Interact To Regulate The Actin Cytoskeleton In Drosophila, Bridget Elsa Varughese

Wayne State University Dissertations

Guidance receptors such as Frazzled affect cell shape and motility by directly or indirectly modulating the cytoskeleton. Fra is particularly needed for the formation of the posterior commissures in a developing Drosophila embryo. The cytoplasmic tyrosine kinase, Abelson Kinase (Abl) enhances the loss of commissures observed in fra mutant. Abl physically interacts with Frazzled to help guide commissural axons across the midline. Furthermore, the loss of commissural axons is only seen when the actin dynamics are perturbed. Abl is also known to regulate actin-dependent processes underlying formation of filopodia, microspikes and membrane ruffles. So, we established a Drosophila S2 cell …

Molecular Mechanisms Of Snare Assembly And Expulsion Of Intravesicular Contents In Cell Secretion, Leah Jiyoung Zhang

Wayne State University Dissertations

For nearly half a century, it was believed that during cell secretion, membrane-bound secretory vesicles completely merge at the cell plasma membrane resulting in the diffusion of intra-vesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. This explanation made no sense or logic, since following cell secretion partially empty vesicles accumulate as demonstrated in electron micrographs. Furthermore, with the `all or none' mechanism of cell secretion by complete merger of secretory vesicle membrane at the cell plasma membrane, the cell is left with little regulation and control of the amount of content release. …

A Novel In Vivo Protein Refolding Technique, Yuefei Huang

Wayne State University Dissertations

Proteins perform their functions in their native folded states and misfolding of proteins may cause severe diseases, including Alzheimer's disease, Parkinson's disease, prion disease and diabetes. Understanding protein folding is important for us to engineer proteins to treat these diseases. For protein therapeutics, large quantities of properly folded and functional proteins are required. The current technology produces recombinant proteins using either eukaryotic or prokaryotic expression system, both of them have major problems that prevent production of large quantities of properly folded and functional human proteins for protein therapeutics.

Although the eukaryotic cells have comprehensive folding machinery that contains chaperones and …

Human Trophoblast Survival And Invasion In The Developing Placenta: Autocrine Regulation By Hbegf, Philip Jessmon

Wayne State University Dissertations

HBEGF is a multifunctional protein in early pregnancy that induces cytotrophoblast (CTB) cell differentiation to an invasive phenotype, protects against apoptosis, and is involved in an autocrine signaling mechanism that leads to its own protein synthesis. CTBs exist in a low O₂ environment during the first 10 weeks of implantation, during which they invade the decidualized uterine stroma. Inhibitors of intracellular signaling pathways demonstrated that at 20% O₂ HBEGF induces an increase in cell migration through the ERK, MAPK14, JNK, or PIK3 pathways downstream of signaling through its ERBB receptors. Also downstream of these four pathways, HBEGF induces …

Destruction Of Biological Tetrapyrrole Macrocycles By Hypochlorous Acid And Its Scavenging By Lycopene, Dhiman Maitra

Wayne State University Dissertations

Hypochlorous acid (HOCl) is a potent oxidant generated by the hemoprotein myeloperoxidase. Although HOCl plays an important role in the innate immune response,sustained high levels of HOCl has been implicated to play a harmful role. In several pathological conditions such as atherosclerosis, endometriosis and sickle cell disease where HOCl is elevated there are reports of significant free iron accumulation. Free iron is toxic since it can lead to the generation of other secondary free radicals such as hydroxyl radical by Fenton reaction. The exact source and mechanism by which the free iron is generated is not clearly understood. This work …

Mechanical Forces And Tumor Cells: Insight Into The Biophysical Aspects Of Cancer Progression, Indrajyoti Indra

Wayne State University Dissertations

Mechanical forces play an important role in the regulation of cellular behavior and physiological processes including adhesion, migration, proliferation, tissue repair, embryogenesis and development. In addition, a number of diseases including cancer, have been linked to changes in cellular and extracellular mechanical properties. However, whether a correlation exists between the progression of cancer towards metastasis and mechanical factors has not been clearly defined. Additionally, how a cell responds to changes in extracellular mechanical cues as it gains metastatic abilities is poorly understood. To address these questions, we have utilized a panel of murine breast cancer cell lines with progressive metastatic. …

Rox1 Function In Dosage Compensation: Structural / Functional Analysis Of A Non-Coding Rna, Ying Kong

Wayne State University Dissertations

roX1 is a long non-coding RNA involved in the chromosome-wide gene regulation that occurs during dosage compensation in Drosophila. Dosage compensation in Drosophila melanogaster occurs by a global two-fold increase of transcription from the single male X chromosome. This essential process compensates for X chromosome monosomy. The male-specific lethal (MSL) complex, containing five proteins, localizes to the male X chromosome and alters chromatin to modify gene expression. roX1 and roX2 RNAs are redundant components of MSL complex that are required for its exclusive X-localization. Recent studies in our lab have revealed a second role of roX RNAs in heterochromatic gene …

Epigenetic Effects Of Benzo(A)Pyrene In Fundulus Heteroclitus And Danio Rerio, Xiefan Fang

Electronic Theses and Dissertations

DNA methylation is one of the epigenetic mechanisms that controls gene expression and is vulnerable to early life environmental toxicant exposures. Our goal was to use two fish models, Fundulus heteroclitus and Danio rerio (zebrafish), to study the benzo(a)pyrene (BaP) effects on DNA methylation status and whether the alterations could contribute to BaP-mediated reproductive and developmental toxicities. Initially, we used Fundulus to study BaP effects on glycine N-methyltransferase (GNMT) expression throughout development. Fundulus embryos were exposed to waterborne BaP at nominally 10 or 100 μg/L and both GNMT mRNA expression and enzyme activity were measured. Quantitative PCR and whole mount …

Stable Expression Of Tuberculosis Vaccine Antigen In Lettuce Chloroplasts, Priya Saikumar Lakshmi

Electronic Theses and Dissertations

Tuberculosis (TB) is caused by Mycobacterium tuberculosis and is one of the leading reasons of death by an infectious bacterial pathogen. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, a potential candidate antigen, ESAT-6 (6 kDa early secretory antigenic target) was fused with cholera toxin B subunit (CTB). Transplastomic lettuce plants were generated expressing these fusion proteins. Site-specific transgene integration into the chloroplast genome was confirmed by polymerase chain reaction and Southern blot analysis. In transplastomic leaves, expression levels of fusion protein (CTB-ESAT6) varied depending upon …

Evolutionary Relationships Among Staphylococci And The Prevention Of Staphylococcus Aureus Nasal Colonization, Ryan Paul Lamers

Electronic Theses and Dissertations

Staphylococcus is a significant cause of human infection and mortality, worldwide. Currently, there are greater than 60 taxa within Staphylococcus, and nearly all are pathogenic. The collective potential for virulence among species of Staphylococcus heightens the overall clinical significance of this genus and argues for a thorough understanding of the evolutionary relationships among species. Within Staphylococcus, aureus is the most common cause of human infection, where nasal carriage of this bacterium is a known risk factor for autoinfection. The predisposition to infection by nasal carriers of S. aureus, and the ease with which strains are transferred between individuals, suggests that …

Synthesis, Characterization And Antibacterial Activity Of Silver Embedded Silica Nanoparticle/Nanogel Formulation, Roseline Menezes

Electronic Theses and Dissertations

The antibacterial property of silver (Ag) has been known since ancient time. It is reported in the literature that silver nanoparticles (AgNPs) exhibit improved antibacterial properties in comparison to silver ions of equivalent metallic Ag concentration. Such improvement in antibacterial activities is due to the high surface area to volume ratio of AgNPs (which facilitates interaction with the bacterial cells), increased release of silver ions and direct intra-cellular uptake of AgNPs leading to localized release of Ag ions. To date, over 300 consumer products containing AgNPs are available in the market and the inventory is rapidly expanding. The antibacterial efficacy …

Expression And Functional Evaluation Of Exendin 4 Fused To Cholera Toxin B Subunit In Tobacco Chloroplast To Treat Type 2 Diabetes, Ramya Nityanandam

Electronic Theses and Dissertations

The prevalence of type 2 diabetes has been steadily increasing around the globe. Glucagon like peptide (GLP-1), a powerful incretin increases insulin secretion in a glucose dependent manner. But GLP-1 is subjected to rapid enzymatic degradation (half-life: 2 min in circulation). The commercially available GLP-1 analog, exenatide has a longer half life with potent insulinotropic effects (about 2.4 hr) which requires cold storage and daily subcutaneous injections. In this study, exendin 4 (EX4), lizard derived GLP-1R agonist, was expressed as cholera toxin B subunit (CTB)-fusion protein in chloroplasts of tobacco to facilitate transmucosal delivery in the gut by utilizing the …