Biochemistry, Biophysics, and Structural Biology Commons^™

Hedgehog Signaling: A Potential Therapeutic Target For Non-Small Cell Lung Cancer, Ma'in Yehya Maitah

Wayne State University Dissertations

The American Cancer Society estimated that 222,520 Americans were diagnosed with lung cancer and 157,300 died of lung cancer in 2010 (Jemal et al. 2009, 225-249;Jemal et al. 2011, 69-90). The clinical outcome of patients diagnosed with non-small cell lung cancer (NSCLC), the major lung cancer sub-types, is very poor, which calls for innovative research for finding novel therapeutic targets and agents for better treatment outcome.

Emerging evidences have suggested that a phenomenon called Epithelial-to-Mesenchymal Transition (EMT), which shares similar molecular characteristics with cancer stem-like cells, contributes to lung cancer treatment failure. In view of the fact that EMT process …

Mne1 Is A Novel Component Of The Mitochondrial Splicing Apparatus Responsible For Processing Of A Cox1 Group I Intron In Yeast, Talina Watts, Oleh Khalimonchuk, Rachel Z. Wolf, Edward M. Turk, Georg Mohr, Dennis R. Winge

Department of Biochemistry: Faculty Publications

Saccharomyces cerevisiae cells lacking Mne1 are deficient in

intron splicing in the gene encoding the Cox1 subunit of cytochrome

oxidase but contain wild-type levels of the bc1 complex.

Thus, Mne1 has no role in splicing of COB introns or expression

of the COB gene. Northern experiments suggest that splicing of

the COX1 aI5β intron is dependent on Mne1 in addition to the

previously known Mrs1, Mss116, Pet54, and Suv3 factors. Processing

of the aI5_ intron is similarly impaired in mne1∆ and

mrs1∆ cells and overexpression of Mrs1 partially restores the

respiratory function of mne1∆ cells. Mrs1 …

Development Of Biomarkers For Endocrine Disrupting Compounds In The Fathead Minnow, Pimephales Promelas, Tara Frare

Undergraduate Review

Organic waste compounds (OWC’s) are an environmental concern that can have adverse effects on humans, animals, and the environment. Ethinyl estradiol (EE2) is one common OWC and is an active ingredient in oral birth control pills. The presence of EE2 in aquatic environments and drinking water can be linked to decreased fertility and altered physiology in fish. I am developing Q-PCR biomarkers to detect changes in fish at low concentrations before adverse effects occur at higher concentrations. RNA was extracted from twenty fathead minnows that had not been exposed to OWC’s and will be used to synthesize cDNA. Thus far …

Method Development And Applications To Screening And Characterization Of Rrna-Targeting Small Molecules, Papa Nii Asare-Okai

Wayne State University Dissertations

A series of single ring aminoglycoside analogues was tested for binding to a model RNA representing the A site using electrospray ionization mass spectrometry (ESI-MS). Several of the synthetic analogues with low molecular weights were found to bind to the RNA with affinities comparable to the parental aminoglycoside neamine, with apparent dissociation constants in the low micromolar range. Salt dependence of the affinity constants for the single ring analogues revealed a predominantly electrostatic binding mode. Footprinting experiments revealed that one of the compounds (DHR23) has a similar binding site as the antibiotic paromomycin. DMS chemical probing results also suggest that …

Mechanisms Of Alpha]-Synuclein-Induced Neurodegenertaion In Parkinson's Disease And Stroke, Cherine Belal

Electronic Theses and Dissertations

Parkinson’s disease (PD) is a debilitating neurodegenerative disorder affecting one million Americans. Despite its social and economic impact, the pathological cascades that lead to neuron dysfunction and degeneration in PD are poorly understood. Endoplasmic reticulum (ER) stress has been implicated as an initiator or contributing factor in neurodegenerative diseases including PD. The ER is an organelle central to protein folding and intracellular Ca2+ homeostasis. Perturbations of these functions result in ER stress and upregulation of ER stress proteins, of which some have been implicated in counteracting ER stress-induced cell death. The mechanisms that lead to ER stress and how ER …

Molecular Details Of The Mitochondrial Iron Sulfur Cluster Assembly Pathway, Swati Rawat

Wayne State University Dissertations

MOLECULAR DETAILS OF THE MITOCHONDRIAL IRON SULFUR CLUSTER ASSEMBLY PATHWAY

Iron-sulfur clusters are an important class of prosthetic group involved in electron transfer, enzyme catalysis, and regulation of gene expression. Their biosynthesis requires complex machinery located within the mitochondrion since free iron and sulfide are extremely toxic to the cell. Defects in this pathway results in several diseases such as Friedreich's Ataxia (FRDA), Sideroblastic Anemia and ISCU Myopathy. Therefore molecular details of the biogenesis pathway will provide deep insight in the pathway and treatment options for these diseases. FRDA is caused by deficiency of a single protein called as `Frataxin'. …

Cam Kinase Regulation Of Akt And Bad In Prostate Cancer Cells, John M. Schmitt, Samantha F. Smith

Faculty Publications - Department of Biological & Molecular Science

Abstract AKT and its substrate BAD promote prostate cancer cell survival. Agonists, such as carbachol, and hormones that increase intracellular calcium concentration can activate AKT leading to cancer cell survival. LNCaP prostate cancer cells express the carbachol-sensitive M3-subtype of GPCR’s that increase intracellular calcium and activate the family of Ca2+/Calmodulin-dependent Protein Kinases (CaM Ks). One type of CaM Kinase, CaM Kinase Kinase (CaM KK), directly phosphorylates AKT on threonine 308. AKT phosphorylation and activation can enhance cell survival through phosphorylation BAD protein and the subsequent blockade of caspase activation. Our goals were to examine the mechanism of carbachol activation of …

Trp Channel Regulation Of Estrogen Signaling, John M. Schmitt, Jessica N. Magill

Faculty Publications - Department of Biological & Molecular Science

Abstract Calcium regulates numerous cell functions including growth and development. Calcium can enter cells through transient receptor potential channels (TRPCs). Previous studies in MCF-7 cells have suggested that the expression of one particular TRPC, TRPC6, correlates with cell transformation and disease progression. Calcium has several cellular targets including the Calcium/Calmodulin-dependent protein kinases (CaM Ks) and ERK. Previous work has shown that estrogen (E2) may utilize CaM Ks and ERK to promote breast cancer cell proliferation, however the possible involvement of TRPCs in this pathway is currently unknown. Our objective was to understand which E2 receptor is used in our system …

Disorder Predictors Also Predict Backbone Dynamics For A Family Of Disordered Proteins, Gary W. Daughdrill, Wade M. Borcherds, Hongwei Wu

Molecular Biosciences Faculty Publications

Several algorithms have been developed that use amino acid sequences to predict whether or not a protein or a region of a protein is disordered. These algorithms make accurate predictions for disordered regions that are 30 amino acids or longer, but it is unclear whether the predictions can be directly related to the backbone dynamics of individual amino acid residues. The nuclear Overhauser effect between the amide nitrogen and hydrogen (NHNOE) provides an unambiguous measure of backbone dynamics at single residue resolution and is an excellent tool for characterizing the dynamic behavior of disordered proteins. In this report, we show …

Dynamic Light Scattering And Zeta Potential Of Colloidal Mixtures Of Amelogenin And Hydroxyapatite In Calcium And Phosphate Rich Ionic Milieus, Vuk Uskoković, Roselyn Odsinada, Sonia Djordjevic, Stefan Habelitz

Pharmacy Faculty Articles and Research

The concept of zeta-potential has been used for more than a century as a basic parameter in controlling the stability of colloidal suspensions, irrespective of the nature of their particulate ingredients – organic or inorganic. There are prospects that self-assembly of peptide species and the protein-mineral interactions related to biomineralization may be controlled using this fundamental physicochemical parameter. In this study, we have analyzed the particle size and zeta-potential of the full-length recombinant human amelogenin (rH174), the main protein of the developing enamel matrix, in the presence of calcium and phosphate ions and hydroxyapatite (HAP) particles. As calcium and phosphate …

Altered Self-Assembly And Apatite Binding Of Amelogenin Induced By N-Terminal Proline Mutation, Li Zhu, Vuk Uskoković, Thuan Le, Pamela Denbesten, Yulei Huang, Stefan Habelitz, Wu Li

Pharmacy Faculty Articles and Research

Objective—A single Pro-70 to Thr (p.P70T) mutation of amelogenin is known to result in hypomineralized amelogenesis imperfecta (AI). This study aims to test the hypothesis that the given mutation affects the self-assembly of amelogenin molecules and impairs their ability to conduct the growth of apatite crystals.

Design—Recombinant human full-length wild-type (rh174) and p.P70T mutated amelogenins were analyzed using dynamic light scattering (DLS), protein quantification assay and atomic force microscopy (AFM) before and after the binding of amelogenins to hydroxyapatite crystals. The crystal growth modulated by both amelogenins in a dynamic titration system was observed using AFM.

Results—As …

Targeted Identification Of Metastasis-Associated Cell-Surface Sialoglycoproteins In Prostate Cancer, Lifang Yang, Julius O. Nyalwidhe, Sigi Guo, Richard R. Drake, O. John Semmes

Bioelectrics Publications

Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC₄ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins …

An Apoptosis Targeted Stimulus With Nanosecond Pulsed Electric Fields (Nspefs) In E4 Squamous Cell Carcinoma, Wei Ren, Stephen J. Beebe

Bioelectrics Publications

Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was [95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels. …

The Proteomic Response Of Sea Squirts (Genus Ciona Congeners) To Heat Stress: Evidence For Differential Thermal Sensitivities, Loredana Serafini

Master's Theses

The sea squirts Ciona intestinalis and C. savignyi have disparate distribution patterns, which may result from differences in their thermal tolerance limits. Because C. intestinalis, an almost cosmopolitan species, has a more widespread distribution, it is thought that it is better adapted to endure a wide range of temperatures. In order to compare the heat stress response between these two congeners, we studied global changes in protein expression, using a proteomics approach. To characterize the response to extreme heat stress, animals of both species were exposed to temperatures of 22°C, 25°C, and 28°C for 6 h, and then were …

Roles Of The 15-Kda Selenoprotein (Sep15) In Redox Homeostasis And Cataract Development Revealed By The Analysis Of Sep 15 Knockout Mice, Marina V. Kasaikina, Dmitri E. Fomenko, Vyacheslav M. Labunskyy, Salil A. Lachke, Wenya Qiu, Juliet A. Moncaster, Jie Zhang, Mark W. Wojnarowicz, Sathish Kumar Natarajan, Mikalai I. Malinouski, Ulrich Schweizer, Petra A. Tsuji, Bradley A. Carlson, Richard L. Maas, Marjorie F. Lou, Lee E. Goldstein, Dolph L. Hatfield, Vadim N. Gladyshev

School of Veterinary and Biomedical Sciences: Faculty Publications

The 15-kDa selenoprotein (Sep15) is a thioredoxin-like, endoplasmic reticulum-resident protein involved in the quality control of glycoprotein folding through its interaction with UDP-glucose:glycoprotein glucosyltransferase. Expression of Sep15 is regulated by dietary selenium and the unfolded protein response, but its specific function is not known. In this study, we developed and characterized Sep15 KO mice by targeted removal of exon 2 of the Sep15 gene coding for the cysteinerich UDP-glucose:glycoprotein glucosyltransferase-binding domain. These KO mice synthesized a mutant mRNA, but the shortened protein product could be detected neither in tissues nor in Sep15 KO embryonic fibroblasts. Sep15 KO mice were viable …

Differential Transcription Of Cytochrome P450s And Glutathione S Transferases In Ddt-Susceptible And -Resistant Drosophila Melanogaster Strains In Response To Ddt And Oxidative Stress, Lijie Sun, Brandi Schemerhorn, Amber Jannasch, Kent R. Walters Jr., Jiri Adamec, William M. Muir, Barry R. Pittendrigh

Department of Biochemistry: Faculty Publications

Metabolic DDT resistance in Drosophila melanogaster has previously been associated with constitutive over-transcription of cytochrome P450s. Increased P450 activity has also been associated with increased oxidative stress. In contrast, over-transcription of glutathione S transferases (GSTs) has been associated with resistance to oxidative stress. However, little is known in regards to the impact of xenobiotics on induction of P450s and GSTs and if there exist differences in inducibility between the pesticide susceptible and resistant strains. Thus, we investigated the transcriptional expression of GSTs and P450s in DDT resistant (Wisconsin) and susceptible (Canton-S) Drosophila strains in response to …

The Impact Of Tricaine Methanesulfonate, 2-Phenoxyethanol, And Carvone-Methyl Salicylate On The Innate Immune Response Of Zebrafish (Danio Rerio), Charles R. Wulff

Honors Theses

Anesthesia plays a vital role in the maintenance of aquaculture species, where it is used to minimize stress during complex handling tasks such as transport, assessment, and harvesting. However, anesthetics have been shown to suppress the innate immune response, which could impact immunity and increase risk of infection. Tricaine methanesulfonate (MS-222) and 2-Phenoxyethanol (2-PE) represent two of the most commonly used anesthetics in aquaculture, with R-(+)-carvone, in the form of carvone-methyl salicylate (CMS) has recently been proposed as an alternative anesthetic for food fish. These three anesthetics were used to assess the influence of anesthetics on the immune system of …

A Cell Electrofusion Microfluidic Device Integrated With 3d Thin-Film Microelectrode Arrays, Ning Hu, Jun Yang, Shizhi Qian, Sang W. Joo, Xiaolin Zheng

Mechanical & Aerospace Engineering Faculty Publications

A microfluidic device integrated with 3D thin film microelectrode arrays wrapped around serpentine-shaped microchannel walls has been designed, fabricated and tested for cell electrofusion. Each microelectrode array has 1015 discrete microelectrodes patterned on each side wall, and the adjacent microelectrodes are separated by coplanar dielectric channel wall. The device was tested to electrofuse K562 cells under a relatively low voltage. Under an AC electric field applied between the pair of the microelectrode arrays, cells are paired at the edge of each discrete microelectrode due to the induced positive dielectrophoresis. Subsequently, electric pulse signals are sequentially applied between the microelectrode arrays …

Thiol Peroxidases Mediate Specific Genome-Wide Regulation Of Gene Expression In Response To Hydrogen Peroxide, Dmitri E. Fomenko, Ahmet Koc, Natalia Agisheva, Michael Jacobsen, Alaattin Kaya, Mikalai Malinouski, Julian C. Rutherford, Kam-Leung Siu, Dong-Yan Jin, Dennis R. Winge, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Hydrogen peroxide is thought to regulate cellular processes by direct oxidation of numerous cellular proteins, whereas antioxidants, most notably thiol peroxidases, are thought to reduce peroxides and inhibit H₂O₂ response. However, thiol peroxidases have also been implicated in activation of transcription factors and signaling. It remains unclear if these enzymes stimulate or inhibit redox regulation and whether this regulation is widespread or limited to a few cellular components. Herein, we found that Saccharomyces cerevisiae cells lacking all eight thiol peroxidases were viable and withstood redox stresses. They transcriptionally responded to various redox treatments, but were unable to …

Differential Precipitation And Solubilization Of Proteins., Barry Ryan

Books/Book Chapters/ Proceedings

Differential protein precipitation is a rapid and economical step in protein purification and is based on exploiting the inherent physico-chemical properties of the polypeptide. Precipitation of recombinant proteins, lysed from the host cell, is commonly used to concentrate the protein of choice before further polishing steps with more selective purification columns (e.g. His-Tag, Size Exclusion etc.). Recombinant proteins can also precipitate naturally as inclusion bodies due to various influences during over-expression in the host cell. Although this phenomenon permits easier initial separation from native proteins, these inclusion bodies must carefully be differentially solubilised so as to reform functional, correctly folded …