Molecular Biology Commons^™

Novel Role Of Erbb-2 In Inhibition Of Jagged-1-Mediated Trans-Activation Of Notch In Breast Cancer, Kinnari Pandya

Dissertations

The ErbB-2 gene is amplified and the resulting protein product overexpressed in 15-30% of breast tumors, and associated with aggressive behavior and poor overall survival. Currently, there are two FDA approved therapies targeting ErbB-2 for the treatment of ErbB-2 positive breast cancer: trastuzumab, a humanized monoclonal antibody is directed against the extracellular domain of ErbB-2 and lapatinib, a dual EGFR/ErbB-2 tyrosine kinase inhibitor. Unfortunately, anti-ErbB-2 therapy resistance remains a major problem in metastatic breast cancer. Our data suggested that gene amplification or overexpression of ErbB-2 inhibits Notch-1 transcriptional activity and trastuzumab or lapatinib increased

Notch-1 transcriptional activity. Furthermore, Notch-1 is …

Significance Of Protein Interactions In Mediating Af9 Function, Bhavna Malik

Dissertations

Rearrangements of the MLL gene at chromosome band 11q23 have been associated with a heterogeneous group of lymphoid, myeloid and mixed lineage leukemias. MLL rearrangements occur approximately in 70% of infant leukemias and are also common in therapy-related leukemias where patients were previously treated with topoisomerase II inhibitors. Unfortunately, these patients have a poor prognosis. MLL gene rearrangements give rise to chimeric proteins that contain the N-terminal portion of MLL fused to the C-terminal portion of over 50 different fusion partners. The chimeric proteins cause constitutive expression of some MLL target genes such as HOXA9 and MEIS1, and enhanced proliferation …

Molecular Functions Of Mll Phd3 Binding To Its Ligands Cyp33 And H3k4me3, Gayathree Raman

Dissertations

Mixed Lineage Leukemia protein (MLL) is required for proper embryonic development, and hematopoiesis. It is a SET domain containing histone methyl transferase that trimethylates histone H3 on lysine 4 (H3K4Me3), a histone modification that correlates with active transcription. The 3rd PHD finger of MLL binds to H3K4me3. Thus MLL is a "writer" with an embedded "reader" for H3K4Me3. Cyp33 is another known ligand of MLL PHD3. Over expression of Cyp33 results in transcriptional repression of MLL target genes.

The aim of this study is to determine the biological function of MLL PHD3 binding to H3K4Me3 or Cyp33. Cyp33 binding to …

Extracellular Proteases Are Key Mediators Of Staphylococcus Aureus Virulence Via The Global Modulation Of Virulence-Determinant Stability, Stacey L. Kolar, J. Antonio Ibarra, Frances E. Rivera, Joe M. Mootz, Jessica E. Davenport, Stanley M. Stevens Jr., Alexander R. Horswill, Lindsey N. Shaw

Molecular Biosciences Faculty Publications

Staphylococcus aureus is a highly virulent and successful pathogen that causes a diverse array of diseases. Recently, an increase of severe infections in healthy subjects has been observed, caused by community-associated methicillin-resistant S. aureus (CA-MRSA). The reason for enhanced CA-MRSA virulence is unclear; however, work suggests that it results from hypersecretion of agr-regulated toxins, including secreted proteases. In this study, we explore the contribution of exo-proteases to CA-MRSA pathogenesis using a mutant lacking all 10 enzymes. We show that they are required for growth in peptide-rich environments, serum, in the presence of antimicrobial peptides (AMPs), and in human blood. …

A Transient Α-Helical Molecular Recognition Element In The Disordered N-Terminus Of The Sgs1 Helicase Is Critical For Chromosome Stability And Binding Of Top3/Rmi1, Jessica A. Kennedy, Gary W. Daughdrill, Kristina H. Schmidt

Molecular Biosciences Faculty Publications

The RecQ-like DNA helicase family is essential for the maintenance of genome stability in all organisms. Sgs1, a member of this family in Saccharomyces cerevisiae, regulates early and late steps of double-strand break repair by homologous recombination. Using nuclear magnetic resonance spectroscopy, we show that the N-terminal 125 residues of Sgs1 are disordered and contain a transient α-helix that extends from residue 25 to 38. Based on the residue-specific knowledge of transient secondary structure, we designed proline mutations to disrupt this α-helix and observed hypersensitivity to DNA damaging agents and increased frequency of genome rearrangements. In vitro binding assays …

Identification Of Proteins At Active, Stalled, And Collapsed Replication Forks Using Isolation Of Proteins On Nascent Dna (Ipond) Coupled With Mass Spectrometry, Bianca M. Sirbu, W. Hayes Mcdonald, Huzefa Dungrawala, Akosua Badu-Nkansah, Gina M. Kavanaugh, Yaoyi Chen, David L. Tabb, David Cortez

Molecular Biosciences Faculty Publications

Both DNA and chromatin need to be duplicated during each cell division cycle. Replication happens in the context of defects in the DNA template and other forms of replication stress that present challenges to both genetic and epigenetic inheritance. The replication machinery is highly regulated by replication stress responses to accomplish this goal. To identify important replication and stress response proteins, we combined isolation of proteins on nascent DNA (iPOND) with quantitative mass spectrometry. We identified 290 proteins enriched on newly replicated DNA at active, stalled, and collapsed replication forks. Approximately 16% of these proteins are known replication or DNA …

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 …

Biology And Ecology Of Glyphosate-Resistant Giant Ragweed, Kabelo Segobye

Open Access Theses

Giant ragweed (Ambrosia trifida L.) is a competitive annual plant found in disturbed landscapes and is the most troublesome weed in Indiana and the US Corn Belt. It is one of the most common and problematic weeds in corn and soybean production. The introduction of herbicide glyphosate, N-(phosphonomethyl) glycine in early 1970's provided farmers with a better and low-cost tool to control weeds. The use of glyphosate drastically increased after the development of glyphosate resistant agronomic crops in 1996 and was use as a post-emergence selective herbicide. This led to overreliance and repeated use of glyphosate for weed control especially …

Primary Pathways Of Intracellular Ca2+ Mobilization By Nanosecond Pulsed Electric Field, Iurii Semenov, Shu Xiao, Andrei G. Pakhomov

Bioelectrics Publications

Permeabilization of cell membranous structures by nanosecond pulsed electric field (nsPEF) triggers transient rise of cytosolic Ca²⁺ concentration ([Ca²⁺]_i), which determines multifarious downstream effects. By using fast ratiometric Ca²⁺ imaging with Fura-2, we quantified the external Ca²⁺ uptake, compared it with Ca²⁺ release from the endoplasmic reticulum (ER), and analyzed the interplay of these processes. We utilized CHO cells which lack voltage-gated Ca²⁺ channels, so that the nsPEF-induced [Ca²⁺]_i changes could be attributed primarily to electroporation. We found that a single 60-ns pulse caused fast [Ca2+]_i increase …

Relationship Of Cross-Linking Potential To Mechanism Of Cell Death, Adam N. Spierer

Honors Theses

Mechlorethamine (HN2), a nitrogen derivative of mustard gas, was the first synthetic anti-tumor chemotherapeutic because it forms covalent cross-links between strands of duplex DNA. HN2 represents a class of bifunctional alkylating agents that are both chemotherapeutic and carcinogenic: diepoxybutane (DEB), the active form of the pro-drug treosulfan, and epichlorohydrin (ECH), a structural hybrid of HN2 and DEB, also form covalent cross-links between DNA. While HN2 and DEB are clinically used as anti-tumor chemotherapeutics, ECH is a structural hybrid of these two compounds not used in a clinical setting. Accordingly, we aimed to understand the relationship between the cross-linking potential of …

Regulation Of The Glycine Transporter 1 (Glyt1) By Pkca-Dependent Ubiquitination, Susana Barrera

Open Access Theses & Dissertations

Glycine is one of the major inhibitory neurotransmitters in the central nervous system. It is implicated in the regulation of motor-sensory function such as; pain perception, reflex responses, and is essential for early development of the CNS. In addition to its inhibitory actions, it acts as an obligatory co-agonist for the activation of glutamatergic NMDARs, which are of pivotal importance in the process of learning and memory, its dysfunction leads to several neurodegenerative diseases. Two high affinity Na+/Cl- dependent transporters GlyT1 and GlyT2 tightly regulate the availability of glycine present at the synapse for the activation of NMDARs and GlyRs. …

Aβ Alters The Dna Methylation Status Of Cell-Fate Genes In An Alzheimer’S Disease Model, Gary D. Isaacs, Noor Taher, Courtney Mckenzie, Rebecca Garrett, Matthew Baker, Nena Fox

Faculty Publications and Presentations

Alzheimer’s disease (AD) is characterized by neurofibrillary tangles and extracellular amyloid-β plaques (Aβ). Despite ongoing research, some ambiguity remains surrounding the role of Aβ in the pathogenesis of this neurodegenerative disease. While several studies have focused on the mutations associated with AD, our understanding of the epigenetic contributions to the disease remains less clear. To that end, we determined the changes in DNA methylation in differentiated human neurons with and without Aβ treatment. We isolated the DNA from neurons treated with Aβ or vehicle, and digested the two samples with either a methylation-sensitive (HpaII) or a methylation-insensitive (MspI) restriction endonuclease. …

Interaction Between Centromeric Histone H3 Variant And Shugoshin, Visarut Buranasudja

Open Access Theses

Precise and faithful segregation of chromosome segregation during mitosis depends on the ability of the cell to regulate chromosome bi-orientation on the mitotic spindle. Shugoshin (Sgo1), the protector of meiotic centromeric cohesin, is required for proper establishment of chromosome bi-orientation. Sgo1 plays a crucial role as part of a mitotic tension sensor between sister chromatids. Recently, Sgo1 has been reported to interact with histone H3 at the pericentromere region, as an important factor for tension sensing and chromosome segregation. However, the role of Sgo1 in tension sensing at centromere is still elusive. The centromere is the region of attachment of …

Investigating The Metal Binding Affinity Of Bacterial Zinc Transporters: Zupt And Truncated Znta (Znta-Pina), Fei Nie

Wayne State University Theses

Zinc is essential trace elements for all living organisms and serves as cofactor and structural cofactors for more than 300 enzymes. ZupT is one of the members of ZIP super family of transporter proteins, which were identified as iron and zinc transporter in Eukaryotes at first. However, recent evidence showed that some of members can also transport other metal ions, such as manganese or cadmium, and ZIP proteins also exist in bacteria. ZupT is responsible for zinc uptake in E. coli; however, its full metal selectivity is not yet characterized. In addition, the mechanism of transport is also unknown. Therefore, …

Effect Of Estrogen On Manganese-Induced Toxicity On Embryonic Astrocytes, Tyler T. Huynh, Kimberly J. Baker, Harold L. Komiskey

PCOM Scholarly Works

Manganese (Mn) is a natural trace metal that is essential for many physiological functions in the human body. Astrocytes in the central nervous system are susceptible reservoirs for Mn accumulation. Estrogen, a steroidal hormone, has been shown to mitigate Mn-induced toxicity in cultures of postnatal astrocytes. However, differences in expression/inducibility of glutamate transporters and glutamine synthetase, transmitters, and the natural gonadal steroids and their receptors are known to occur in astrocyte cultures derived from various stages of fetal and postnatal development. Cultures of embryonic (E18) hippocampal astrocytes were examined in this study for the ability of 17 β-estradiol (E2) to …

The Genetic Engineering Of Motor Proteins, Rachael M. Hartz

Legacy Theses & Dissertations (2009 - 2024)

Molecular motors are a remarkable feature within living organisms that are responsible for directional mechanical motion, which is driven by adenosine triphosphate (ATP) hydrolysis. Actin-binding molecular motors are of specific interest in the field of nanotechnology as filamentous actin is capable of carrying cargo, such as quantum dots, while it is translocated along a motor coated surface. The binding regions of motor proteins, which are known to interact with actin, such as Myosin, have been thoroughly examined and identified. Rapid genetic engineering of the ATP-hydrolyzing enzyme, adenosine kinase, to incorporate these binding regions is possible through the use of site- …

Mutational Analysis Of An Rna Aptamer, Deblina Biswas

Legacy Theses & Dissertations (2009 - 2024)

Aptamers are oligonucleotide molecules (DNA or RNA) that are selected against specific target proteins, nucleic acids or small molecules by using the well established technique of SELEX (Systemic Evolution of Ligands by Exponential Enrichment). Their ability to bind with high affinity and specificity has opened up a wide arena of scientific applications. Aptamers have been shown to have many potential therapeutic and bioanalytical contributions such as in the treatment of diseases like cancer, cardiovascular and other major ailments. The aim of this dissertation is to study and analyze double transversion involving 4 bases in a RNA aptamer molecule: MinB4-au-flipS clamp …

Role Of The Endocannabinoid System In Cardiac Remodeling, Monica Gole

Electronic Theses and Dissertations

The endocannabinoid system and CB1 and CB2 cannabinoid receptors have been implicated in cardiac remodeling with CB1 inhibition and CB2 activation reported to be beneficial. However, the role of these receptors in cardiac remodeling secondary to hypertension is not known. Moreover, the functions of the CB1 and CB2 cannabinoid receptors in cardiac ventricular fibroblasts and their downstream fibrotic pathways has not yet been elucidated. This study was designed to determine the temporal myocardial expression pattern of CB1 and CB2 receptors in cardiac remodeling secondary to pressure overload (PO) induced hypertension at 3, 5, 14 and 28 days. The expression of …

The Mononuclear Metal Center Of Type-I Dihydroorotase From Aquifex Aeolicus, Brian Fp Edwards, Roshini Fernando, Philip D. Martin, Edward Grimley, Melissa Cordes, Asmita Vaishnav, Joseph S. Brunzelle, Hedeel Evans, David R. Evans

Wayne State University Associated BioMed Central Scholarship

Abstract

Background

Dihydroorotase (DHO) is a zinc metalloenzyme, although the number of active site zinc ions has been controversial. E. coli DHO was initially thought to have a mononuclear metal center, but the subsequent X-ray structure clearly showed two zinc ions, α and β, at the catalytic site. Aquifex aeolicus DHO, is a dodecamer comprised of six DHO and six aspartate transcarbamoylase (ATC) subunits. The isolated DHO monomer, which lacks catalytic activity, has an intact α-site and conserved β-site ligands, but the geometry of the second metal binding site is completely disrupted. However, the putative β-site is restored when the …

Disulfide By Design 2.0: A Web-Based Tool For Disulfide Engineering In Proteins, Douglas B. Craig, Alan A. Dombkowski

Wayne State University Associated BioMed Central Scholarship

Abstract

Background

Disulfide engineering is an important biotechnological tool that has advanced a wide range of research. The introduction of novel disulfide bonds into proteins has been used extensively to improve protein stability, modify functional characteristics, and to assist in the study of protein dynamics. Successful use of this technology is greatly enhanced by software that can predict pairs of residues that will likely form a disulfide bond if mutated to cysteines.

Results

We had previously developed and distributed software for this purpose: Disulfide by Design (DbD). The original DbD program has been widely used; however, it has a number …