Biochemistry, Biophysics, and Structural Biology Commons^™

The Second Generation Proteasome Inhibitor Carfilzomib Interacts Synergistically With Hdac Inhibitors In Diffuse Large B-Cell Lymphoma Cells Through Multiple Mechanisms And Circumvents Bortezomib Resistance, Dmitry Lembersky

Theses and Dissertations

Mechanisms underlying the interactions between the proteasome inhibitor carfilzomib and HDAC inhibitors were examined in both germinal center (GC) and activated B-cell (ABC) subtypes of human diffuse large B-cell lymphoma (DLBCL). Simultaneous exposure to minimally toxic concentrations of carfilzomib and HDAC inhibitor vorinostat resulted in the release of mitochondrial pro-apoptotic proteins SMAC and cytochrome c, pro-apoptotic caspase activation, and synergistic induction in apoptosis in both ABC and GC DLBCL subtypes. These events were associated with a marked increase in the stress kinase JNK, ROS generation, G2-M cell cycle arrest, as well as induction of DNA damage. Genetic knockdown of JNK …

The Role Of Brca1 In Dna Double-Strand Break Repair, Seth Dever

Theses and Dissertations

Mutations in the breast cancer susceptibility 1 (BRCA1) gene are linked to breast as well as ovarian cancers. However, most cancer-causing mutations within the BRCA1 gene have been found in the N’ and C’ terminal regions of the BRCA1 protein, both believed to be important for DNA double-strand break (DSB) repair. The BRCA1 C’ terminal (BRCT) repeats have been implicated in phospho-serine protein binding whereas the N’ terminal RING domain interacts with the BARD1 protein to form a hetero-dimeric complex with E3 ubiquitin ligase activity. The BRCA1 BRCT domain binds CtIP, BACH1, and RAP80, all of which have been directly …

Scribble As A Possible Binding Partner For The Map Kinases Erk2 And Erk6, William Allen

Theses and Dissertations

We worked on finding a new kinase regulator to develop basic data to be used for cancer prevention. Our work found a link between three previously unrelated proteins involved in cancer, ERK2 and ERK6 and Scribble. The MAP kinase cascade is involved in cell proliferation, which is highly deregulated in cancer. Through the screening of ERK6 associating molecules, we found the cell polarity, and cell cycle related molecule Scribble. Furthermore, we found that Scribble was a dual-specific kinase regulator. We clearly demonstrated that these ERKs interact with Scribble through the LRR and the PDZ domains of Scribble. We hypothesize that …

Ionic Selectivity In L-Type Calcium Channels By Electrostatics And Hard-Core Repulsion, Dezso Boda, Douglas Henderson, Monika Valisko, Bob Eisenberg, Dirk Gillespie

Faculty Publications

A physical model of selective "ion binding" in the L-type calcium channel is constructed, and consequences of the model are compared with experimental data. This reduced model treats only ions and the carboxylate oxygens of the EEEE locus explicitly and restricts interactions to hard-core repulsion and ion–ion and ion–dielectric electrostatic forces. The structural atoms provide a flexible environment for passing cations, thus resulting in a self-organized induced-fit model of the selectivity filter. Experimental conditions involving binary mixtures of alkali and/or alkaline earth metal ions are computed using equilibrium Monte Carlo simulations in the grand canonical ensemble. The model pore rejects …

The Zinc Finger Protein Zn72d And Dead Box Helicase Belle Interact And Control Maleless Mrna And Protein Levels, Kathleen A. Worringer, Feixia Chu, Barbara Panning

Molecular, Cellular & Biomedical Sciences

Background: The Male Specific Lethal (MSL) complex is enriched on the single X chromosome in male Drosophila cells and functions to upregulate X-linked gene expression and equalize X-linked gene dosage with XX females. The zinc finger protein Zn72D is required for productive splicing of the maleless (mle) transcript, which encodes an essential subunit of the MSL complex. In the absence of Zn72D, MLE levels are decreased, and as a result, the MSL complex no longer localizes to the X chromosome and dosage compensation is disrupted. To understand the Molecular basis of Zn72D function, we identified proteins that interact with Zn72D. …

Consequences Of Hyaluronan Metabolism On Cell Cycle Progression In Prostate Tumor Growth And Metastasis, Alamelu G. Bharadwaj

Department of Biochemistry: Dissertations, Theses, and Student Research

Progression of Prostate cancer (CaP), depends on a complex series of interactions between the tumor and extracellular matrix (ECM) leading to tumor growth and metastasis. Hyaluronan (HA), an ECM component, is elevated in CaP and its accumulation in the tumor microenvironment is dependent on its synthetic enzyme, hyaluronan synthase (HAS), and turnover enzyme, hyaluronidase (Hyal). Although Hyal1 expression can independently prognosticate CaP, and HAS expression shows increase in aggressive prostate cancer cells, the functional relevance of this correlation is unexplored. In these studies we aim to dissect the roles of HAS and Hyal1 in prostate cancer.

Stably overexpressed hyaluronan synthases …

Synthesis Of 2,4,5-Triaminocyclohexane Carboxylic Acid As A Novel 2-Deoxystreptamine Mimetic, Sarah Elizabeth Roberts

Theses and Dissertations

RNAs have become increasingly recognized as possible drug targets due to their involvement in important biochemical functions, as well as their unique but well-defined structures. Recently published crystal structures depict the binding of a series of aminoglycosides- or more specifically- 2-deoxystretamine (2-DOS), the most preserved central scaffold of aminoglycosides, to a conserved 5'-GU-3'region on their target RNAs. A novel unnatural γ-amino acid, 1, has been synthesized using 2-deoxystreptamine as a template through structure-based rational design. The unnatural amino acid has been designed to replace a glycosidic linkage with an amide bond, which may limit the promiscuous binding characteristics of aminoglycosides …

Novel Mechanisms Regulating Cytokine-Induced Gene Expression In Astrocytes And Glioblastoma Cells, Lauren Bryan

Theses and Dissertations

Chronic inflammation in the brain results in the development of several CNS diseases, including Alzheimer’s and Parkinson’s diseases, multiple sclerosis, and tumors. IL-1, a pro-inflammatory cytokine released by activated microglia and astrocytes, instigates the expression of factors promoting the progression of these CNS disorders, including cytokines, chemokines, and components of matrix remodeling systems, such as the plasminogen activator system. IL-1 also increases the mRNA expression and activity of SphK, the enzyme that phosphorylates Sph to form S1P, a bio-active sphingolipid. This thesis demonstrates that IL-1 and S1P enhance the mRNA and protein expression of PAI-1 and uPAR, two key components …

Molecular Mechanisms For Regulation Of Gene Expression By Lysophosphatidic Acid In Ovarian Carcinoma Cells, Regina Oyesanya

Theses and Dissertations

Lysophosphatidic acid (LPA) is a potent bioactive phospholipid mediator that functions through multiple G protein couple receptors (GPCRs). LPA is elevated in ascites of ovarian cancer patients and is involved in growth, survival and metastasis of ovarian cancer cells. Gene promoter analyses revealed that some LPA-target genes share similar sets of binding sites for prominent transcription factors posing the possibility of a general mechanism for activation of their expression by LPA. Detailed investigation of the mechanisms of regulation of cyclooxygenase 2 (Cox-2), a paradigm of LPA-regulated genes, showed that LPA robustly upregulated the expression of Cox-2 in ovarian cancer cells …

Knockdown Of Limiting-Co2–Induced Gene Hla3 Decreases Hco3− Transport And Photosynthetic Ci Affinity In Chlamydomonas Reinhardtii, Deqiang Duanmu, Amy R. Miller, Kempton M. Horken, Donald P. Weeks, Martin H. Spalding

Department of Biochemistry: Faculty Publications

The CO₂-concentrating mechanism (CCM) of Chlamydomonas reinhardtii and other microalgal species is essential for photosynthetic growth in most natural settings. A great deal has been learned regarding the CCM in cyanobacteria, including identification of inorganic carbon (Ci; CO₂ and HCO₃⁻) transporters; however, specific knowledge of analogous transporters has remained elusive in eukaryotic microalgae such as C. reinhardtii. Here we investigated whether the limiting-CO₂–inducible, putative ABC-type transporter HLA3 might function as a HCO₃⁻ transporter by evaluating the effect of pH on growth, photosynthetic Ci affinity, and [¹⁴C]-Ci uptake in …

Role Of Molecular Chaperones In G Protein B5-Regulator Of G Protein Signaling Dimer Assembly And G Protein By Dimer Specificity, Alyson Cerny Howlett

Theses and Dissertations

In order for G protein signaling to occur, the G protein heterotrimer must be assembled from its nascent polypeptides. The most difficult step in this process is the formation of the Gβγ dimer from the free subunits since both are unstable in the absence of the other. Recent studies have shown that phosducin-like protein (PhLP1) works as a co-chaperone with the cytosolic chaperonin complex (CCT) to fold Gβ and mediate its interaction with Gγ. However, these studies did not address questions concerning the scope of PhLP1 and CCT-mediated Gβγ assembly, which are important questions given that there are four Gβs …

Modeling Time-Series Absorbance Spectra, Amarillis Andrea Cornejo, Mitch Menzmer

Capstone Research Projects

The progress of many reactions can be followed by taking time-series ultraviolet-visible (UV-vis) absorbance spectra. Important information about a mechanism can be obtained from such series. Given experimental time-series absorbance spectra and a proposed mechanism, a method for simulating timeseries absorbance spectra using the computer program Chemical Kinetics Simulator (CKS) and a spreadsheet is proposed. Time series absorbance spectra of the reaction of 1x10-4 M chenodeoxycholic acid in 74.7% (w/w) H2SO4/H20 at 60.0 °C was used as the test case. This method is expected to be useful in the evaluation of mechanisms derived from time-series absorbance spectra.

An Attempted Synthesis Of A Menadione Derivative Via A Diels-Alder Reaction, Charity A. Peñaloza

Capstone Research Projects

With menadione and cyclopentadiene as starting materials, a menadione-cyclopentadiene adduct was synthesized via a Diels-Alder reaction. This reaction serves as the first step in a pathway to synthesize a menadione-safranal derivative that could potentially have antioxidant activity.

Phase-Transfer Catalyzed Synthesis Of The Soy Isoflavanoid S-Equol, A. W. Butler, A. L. Calvert, Michael A. Christiansen, M. B. Andrus

Chemistry and Biochemistry Faculty Presentations

S-Equol is an isoflavanoid metabolized from the isoflavone daidzein by intestinal bacteria. Only 30-50 percent of all individuals possess bacteria that produce S-Equol. Recent data supports the compound’s pharmaceutical potential for treating prostate cancer and post-menopausal estrogen deficiency (ref. 1). Because the compound is only minutely available from natural sources, an affordable asymmetric total synthesis would be necessary to generate sufficient quantities for expanded research. Only one asymmetric total synthesis of S-Equol has been reported to date, using a chiral auxiliary to give product in a 10 percent yield over 6 steps (ref. 2). It is our goal to develop …

Synthesis Of Kurasoin B Analogs, J. R. Nielson, Michael A. Christiansen, M. B. Andrus

Chemistry and Biochemistry Faculty Presentations

Kurasoin B (1), isolated in 1996 by Uchida et al, selectively inhibits Farnesyltransferase (FTase), an enzyme responsible for activating human RAS proteins (ref. 1). When RAS proteins are mutated and then activated, they contribute 20 to 30 percent of all human tumors, including those of the pancreas, colon, small intestine, lung, prostate, liver, skin, and thyroid, as well as multiple myeloma and a number of leukemias (ref. 2). Kurasoin B, therefore, has great potential as a cancer drug lead.

Polyglutamine Disruption Of The Huntingtin Exon 1 N Terminus Triggers A Complex Aggregation Mechanism, Ashwani K. Thakur, Murali Jayaraman, Rakesh Mishra, Monika Thakur, Veronique M. Chellgren, In-Ja L Byeon, Dalaver H. Anjum, Ravindra Kodali, Trevor P. Creamer, James F. Conway, Angela M. Gronenborn, Ronald Wetzel

Molecular and Cellular Biochemistry Faculty Publications

Simple polyglutamine (polyQ) peptides aggregate in vitro via a nucleated growth pathway directly yielding amyloid-like aggregates. We show here that the 17-amino-acid flanking sequence (HTT^NT) N-terminal to the polyQ in the toxic huntingtin exon 1 fragment imparts onto this peptide a complex alternative aggregation mechanism. In isolation, the HTT^NT peptide is a compact coil that resists aggregation. When polyQ is fused to this sequence, it induces in HTT^NT, in a repeat-length dependent fashion, a more extended conformation that greatly enhances its aggregation into globular oligomers with HTT^NT cores and exposed polyQ. In a second …

Characterization Of Antibiotic Resistance In Vibrio Cholerae Isolated From Ships' Ballast And Other Environmental Sources, Frank Kornegay Thomson Iii

OES Theses and Dissertations

Ships' ballast has been implicated as a vector in the dispersal of invasive species into new environments. Numerous cases have been documented for the introduction of macroorganisms via ballasting operations, but what is less known is the role of ballast water and residuals in the spread of potentially harmful microbes, especially with reference to novel genotypes. Of particular interest are the genes encoding for various forms of antibiotic resistance, many of which are carried on mobile genetic elements. A four-year sampling effort has yielded over 300 putative isolates of Vibrio cholerae from ships' ballast tanks and various environmental sources, of …

Comparison Of Immune Correlates, Age Related Changes And Proteomic Profiling Of Healthy Individuals Receiving Influenza Vaccines, Gaurav Basu

Theses and Dissertations in Biomedical Sciences

The burden of influenza related infections is substantial, both in terms of illness, lives lost and economic impact on society. The degree of impact of influenza related infections is much higher in the elderly population where it is a leading cause of catastrophic disability; greatly affecting the quality of life of elderly persons above 65 years of age. Vaccination is the mainstay for control and prevention of influenza infections. There are two vaccine formulations that are licensed for use at present. The inactivated influenza vaccines (TIIV) which have been used for 60 years in all age groups and the new …

Role Of The Human Cytomegalovirus Ie2 Protein On Transcriptional Regulation Of Viral Promoters, Siabhon Michelle Harris

Theses and Dissertations in Biomedical Sciences

Human cytomegalovirus (HCMV) is a ubiquitous pathogen able to cause severe mortality and morbidity in immuno-compromised individuals. Successful infection by HCMV is dependent on expression of viral genes essential for replication. Immediate early (IE) gene products are the first subset of viral genes to be expressed during infection and function as key transcriptional regulators. IE2 is one the most predominantly expressed IE proteins and is essential for HCMV infection. IE2 transactivates several viral promoters, including those of the essential viral DNA polymerase (UL54) and UL112-113 gene regions. IE2 is also able to autoregulate is own expression and repress expression of …

Study Of The Dna Damage Complexes Within The Htlv-1 Tax Oncoprotein Interactome, Sidi Mehdi Belgnaoui

Theses and Dissertations in Biomedical Sciences

Human T-cell leukemia virus type 1 (HTLV-1) is a transforming retrovirus that can give rise to adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Tax is a virally encoded oncoprotein that is involved in HTLV-1 mediated cellular transformation. It has been hypothesized that Tax induces genomic instability via repression of the cellular DNA damage repair response. Our laboratory has previously shown that the interaction between Tax and various proteins involved in the DNA-damage response pathway impairs the ability of these proteins to mount an efficient repair response. As part of these observations, we proposed that Tax induces …

Interdependent Regulation Of Cytomegalovirus Proteins In Complex, Lisa L. Bolin

Theses and Dissertations in Biomedical Sciences

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that causes significant morbidity and mortality in the immune compromised. Using the mutine cytomegalovirus (MCMV) model, we identified two genes, M140 and M141, which are determinants of macrophage tropism. Monocytes/macrophages are a critical cell type for cytomegalovirus (CMV) pathogenesis as sites of viral latency and by supporting viral replication and disseminating virus throughout the body. We previously found that the localization of the M140/M141 gene products (pM140/pM141) is different when the proteins are co-expressed as compared to their individual expression. When the proteins are individually expressed pM141 localizes diffusely throughout the cytoplasm and, …

Phosphorylation Of The Estrogen Receptor Alpha (Erα) At Serine 118 By Phospho-P44/42 Mapk And Regulation By Estrogen And Progesterone In Human Uterine Leiomyoma Tissue And Cells, Tonia Lakisha Hermon

Theses and Dissertations in Biomedical Sciences

It is thought that the growth of uterine leiomyomas may be mediated by the interaction of estrogen receptor alpha (ERα) and growth factor pathways and that phosphorylation of ERα at serine 118 (ERα-phospho-Ser118) is important in this interaction. In tissue, immunoblotting and immunohistochemistry were used to investigate the expression of ERα-phospho-Ser118, phosphorylated p44/42 mitogen-activated protein kinase (phospho-p44/42 MAPK), and proliferating cell nuclear antigen (PCNA) in human leiomyoma and myometrial tissues during the proliferative and secretory phases of the menstrual cycle. In vitro studies to assess proliferation of uterine leiomyoma (UtLM) and uterine smooth muscle (UtSMC) cells and expression of ERα-phospho-Ser118 …

A Capillary Electrophoresis Method For Evaluation Of Abeta Proteolysis In Vitro, Benjamin J. Alper, Walter K. Schmidt

Chemistry & Physics Faculty Publications

According to the amyloid hypothesis, Aβ peptides are neurotoxic and underlie development and progression of Alzheimer’s disease (AD). Multiple Aβ clearance mechanisms, including destruction of the peptides by proteolytic enzymes, are hypothesized to regulate physiological Aβ peptide levels. The insulin-degrading enzyme (IDE) is considered one of the predominant enzymes having Aβ degrading activity. Despite its putative role in protecting against AD, relatively few methods exist for studying IDE activity in vitro. We report the application of capillary electrophoresis (CE) as a novel method for evaluating IDE-mediated Aβ 1–40 proteolysis. This method employs chemically unmodified substrates that are readily obtained …

Light-Regulated Sampling Of Protein Kinase Activity (Ii), Collaborative Project

Dyson College- Seidenberg School of CSIS : Collaborative Projects and Presentations

This entry adheres to the use of the quad chart template to provide a succinct description only of the current research project undertaken by the participants. It provides for the following information

1. Participants and Affiliations
2. Overall Project Goals
3. Illustrative picture
4. Specific research/artistic/pedagogic foci

Functionalization, Characterization, And Applications Of Diamond Particles, Modification Of Planar Silicon, And Chemoetrics Analysis Of Ms Data, Li Yang

Theses and Dissertations

In spite of the stablility (lack of reactivity) of diamond powder, I have developed a method for tethering alkyl chains and polymers to deuterium/hydrogen-terminated diamond. One method is through ether linkages via thermolysis of di-tert-amyl peroxide (DTAP). This reaction with DTAP has also been applied to grow polymers on the diamond surface. The other method is atom transfer radical polymerization (ATRP), which was applied to grow polystyrene at the surface of diamond. Both polystyrene-modified diamond and sulfonated polystyrene-modified diamond can be prepared by either method, and can be used for solid phase extraction. Diamond stationary phases are stable under basic …

The First Attachment And Post-Functionalization Of Polybutadiene And Thio-Click Functionalized Polybutadiene On H-Terminated Si(111), Todd Devere Wickard

Theses and Dissertations

I report the attachment of polymers with pendant vinyl groups to hydrogen-terminated silicon(111) (Si(111)-H) under mild conditions. 1,2-addition polybutadiene (Mw 3200-3500) was attached to Si(111)-H at room temperature with visible light. I also report the partial functionalization, in solution, of 1,2-addition polybutadiene with various thiols using thiol-click chemistry. These compounds bind to Si(111)-H via visible light activation. The partially functionalized polybutadienes allow further functionalization at the surface through unreacted carbon-carbon double bonds. Surfaces were characterized with contact angle goniometry, spectroscopic ellipsometry, X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and atomic force microscopy (AFM).

Catena-Poly[[(Pyridine-Κn)Copper(Ii)]-Μ3-Pyridine-2,6-Dicarboxylato-Κ3o2:O2′,N,O6:O6′], Manoj Trivedi, Daya Pandey, Nigam Rath

Chemistry & Biochemistry Faculty Works

In the title compound, [Cu(C7H3NO4)(C5H5N)]n, the CuII atom is in a slightly distorted octa­hedral coordination environment. Each CuII atom is bound to two N atoms and one O atom of the pyridine­dicarboxyl­ate (PDA) ligand in a tridentate manner, one N atom of the pyridine mol­ecule and two bridging carboxyl­ate O atoms of adjacent PDA ligands, leading to a linear one-dimensional chain running along the c axis. These chains are further assembled via weak C-H...O and [pi]-[pi] inter­actions into a three-dimensional supra­molecular network structure. The centroid-centroid distance between the [pi]-[pi] inter­acting pyridine rings is 3.9104 (13) Å. The two N atoms …

Resampling And Editing Of Mischarged Trna Prior To Translation Elongation, Jiqiang Ling, Byung Ran So, Srujana S. Yadavalli, Hervé Roy, Shinichiro Shoji, Kurt Fredrick, Karin Musier-Forsyth, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Faithful translation of the genetic code depends on the GTPase EF-Tu delivering correctly charged aminoacyl-tRNAs to the ribosome for pairing with cognate codons. The accurate coupling of cognate amino acids and tRNAs by the aminoacyl-tRNA synthetases is achieved through a combination of substrate specificity and product editing. Once released by aminoacyl-tRNA synthetases, both cognate and near-cognate aminoacyl-tRNAs were considered to be committed to ribosomal protein synthesis through their association with EF-Tu. Here we show instead that aminoacyl-tRNAs in ternary complex with EF-Tu•GTP can readily dissociate and rebind to aminoacyl-tRNA synthetases. For mischarged species, this allows resampling by the product editing …

Pathogenic Adaptation Of Intracellular Bacteria By Rewiring A Cis-Regulatory Input Function, Suzanne E. Osborne, Don Walthers, Ana M. Tomljenovic, David T. Mulder, Uma Silphaduang, Nancy Duong, Michael J. Lowden

Dartmouth Scholarship

The acquisition of DNA by horizontal gene transfer enables bacteria to adapt to previously unexploited ecological niches. Although horizontal gene transfer and mutation of protein-coding sequences are well-recognized forms of pathogen evolution, the evolutionary significance of cis-regulatory mutations in creating phenotypic diversity through altered transcriptional outputs is not known. We show the significance of regulatory mutation for pathogen evolution by mapping and then rewiring a cis-regulatory module controlling a gene required for murine typhoid. Acquisition of a binding site for the Salmonella pathogenicity island-2 regulator, SsrB, enabled the srfN gene, ancestral to the Salmonella genus, to play a …

Y27632, A Rho-Activated Kinase Inhibitor, Normalizes Dysregulation In Alpha1-Adrenergic Receptor-Induced Contraction Of Lyon Hypertensive Rat Artery Smooth Muscle., Maria Regina Freitas, Masumi Eto, Jason A Kirkbride, Christa Schott, Jean Sassard, Jean-Claude Stoclet

Department of Molecular Physiology and Biophysics Faculty Papers

RhoA-activated kinase (ROK) is involved in the disorders of smooth muscle contraction found in hypertension model animals and patients. We examined whether the alpha1-adrenergic receptor agonist-induced ROK signal is perturbed in resistance small mesentery artery (SMA) of Lyon genetically hypertensive (LH) rats, using a ROK antagonist, Y27632. Smooth muscle strips of SMA and aorta were isolated from LH and Lyon normotensive (LN) rats. After Ca(2+)-depletion and pre-treatment with phenylephrine (PE), smooth muscle contraction was induced by serial additions of CaCl(2). In LH SMA Ca(2+) permeated cells to a lesser extent as compared with LN SMA, while CaCl(2)-induced contraction of LH …