Biochemistry, Biophysics, and Structural Biology Commons^™

Characterization Of Genes Involved In Phycobiliprotein Biosynthesis In Fremyella Diplosiphon And Thermosynechococcus Elongatus, Christina M. Kronfel

LSU New Orleans Theses and Dissertations

Cyanobacteria are photosynthetic organisms that efficiently capture light by utilizing the light-harvesting complexes called phycobilisomes. In many cyanobacteria, phycobilisomes are composed of an allophycocyanin core with phycocyanin and phycoerythrin (PE) rods radiating from the core. These phycobiliproteins have multiple bilin chromophores, such as phycoerythrobilin (PEB), covalently attached to specific cysteine (Cys) residues for efficient photosynthetic light capture. Chromophore ligation on phycobiliprotein subunits occurs through bilin lyase catalyzed reactions.

This study mainly focuses on characterizing the roles of enzymes that are involved in the biosynthetic pathway of the phycobiliproteins within two cyanobacteria Thermosynechococcus elongatus and Fremyella diplosiphon. A combination of molecular …

B7h6: A Cancer Biomarker For The Development Of Novel Immunotherapy Approaches, Mariana Phillips

Seton Hall University Dissertations and Theses (ETDs)

Cancer-based immunotherapy has led the evolution of biologics that can stimulate immune responses towards tumor eradication. The synthesis of small to intermediate size molecules with the targeting and effector functions of mAb may represent a novel class of immunotherapeutics that may overcome the limitations of their biological counterparts.Towards this objective, B7H6 has been identified as a protein ligand localized on the cell surface of transformed tumor cells. B7H6 binds specifically to the activating receptor NKp30, constitutively expressed on all resting and active NK cells. Upon ligand:receptor binding, B7H6 triggers NK cell activation and release of chemokines and pro-inflammatory cytokines such …

Cloning, Purification, And Biochemical Characterization Of Human Prolyl Endopeptidase, Travis K. Moore

Electronic Theses and Dissertations

Eurygaster integriceps Puton, common name sunn Pest, is one of the primary sources of wheat crop wastes in North Africa, Middle East, and Eastern Europe. It feeds by injecting the wheat grain with an enzyme characterized as prolyl endoprotease (spPEP) that breaks down Gluten, the wheat’s main constitutive protein necessary for bread production (Darkoh et al., 2010). Previously, it has been shown that peptides isolated from Lactobacillus hydrolysates of caseins in bovine milk are able to inhibit mammalian PEP in colon cells, as well as bacterial PEP (Juillerat-Jeanneret et al., 2010). While recombinant versions of these peptides are also potential …

Validation Of The Pre-B Cell Receptor As A Therapeutic Target In B Cell Precursor Acute Lymphoblastic Leukemia, Michael Frank Erasmus

Biomedical Sciences ETDs

This dissertation is built upon the fundamental idea that the pre-B cell receptor (pre-BCR) is important to leukemia cell survival and a logical therapeutic target in B cell precursor acute lymphoblastic leukemia (BCP-ALL). The pre-BCR is expressed early at a specific stage during B cell development where it plays a central role in survival of healthy B lymphocytes. This receptor is composed of the membrane heavy chain (mIgμ) associated with surrogate light chain components, 5 and VpreB. Through the use of advanced imaging modalities, in particular two-color single particle tracking (SPT), we showed that pre-BCRs formed transient, homotypic interactions. These …

Microbal Strans And Methods Of Making And Using, Nicole Roswitha Buan Murphy, Jennifer Catlett

Department of Biochemistry: Faculty Publications

Microbial strains are provided, as are methods of making and using Such microbial strains.

The Role Of Estrogen In Mouse Gastric Stem Cell Homeostasis, Aysha Mohamed Yusuf Alkaabi

Theses

To maintain cellular homeostasis, the epithelial lining of the stomach wall continuously fluctuates between cellular proliferation, differentiation, and apoptosis. A key player in this process is the gastric stem cell (GSC). GSCs are located in the isthmus region of the corpus gastric gland and have the potential to proliferate and differentiate. Although several pathways have been identified to regulate stem cell role in several body tissues, little is known about controlling GSC homeostasis. This project aims to study the role of estrogen (E2) in GSC homeostasis using the well-established mouse gastric epithelial progenitor (mGEP) cell line. Our data showed that …

Pneb193-Derived Suicide Plasmids For Gene Deletion And Protein Expression In The Methane-Producing Archaeon, Methanosarcina Acetivorans, Mitchell T. Shea, Mary E. Walter, Nikolas Duszenko, Anne-Lise Ducluzeau, Jared Aldridge, Shannon K. King, Nicole R. Buan

Department of Biochemistry: Faculty Publications

Gene deletion and protein expression are cornerstone procedures for studying metabolism in any organism, including methane-producing archaea (methanogens). Methanogens produce coenzymes and cofactors not found in most bacteria, therefore it is sometimes necessary to express and purify methanogen proteins from the natural host. Protein expression in the native organism is also useful when studying post-translational modifications and their effect on gene expression or enzyme activity. We have created several new suicide plasmids to complement existing genetic tools for use in the methanogen, Methanosarcina acetivorans. The new plasmids are derived from the commercially available E. coli plasmid, pNEB193, and cannot replicate …

Engineering Fret Biosensors For Microrna Presence/Absence Analysis, Nicholas E. Larkey, Sean M. Burrows

Biomedical Engineering Western Regional Conference

No abstract provided.

An Assessment Of Potential False Positive E.Coli Pyroprints In The Cplop Database, Skyler A. Gordon

Master's Theses

The genetic information found in each species of organism is unique, and can be used as a tool to differentiate at the molecular level. This has caused rapid genotyping methods to become the cornerstone of a new area of research dependent on reading the genome as a form of identification. One of these specific identification methods, known as pyroprinting, relies on the small variation of DNA sequences within the same species to develop a unique, reproducible fingerprint. By simultaneously pyrosequencing multiple polymorphic loci within the ribosomal operons known as the intergenic transcribed spacers, a reproducible output is obtained, known as …

Deuterateer: A Tool For Quantifying Peptide Isotope Precision And Kinetic Proteomics, Bradley C. Naylor, Michael T. Porter, Elise Wilson, Adam Herring, Spencer Lofthouse, Austin Hannemann, Stephen R. Piccolo, Alan L. Rockwood, John C. Price

Faculty Publications

Motivation

Using mass spectrometry to measure the concentration and turnover of the individual proteins in a proteome, enables the calculation of individual synthesis and degradation rates for each protein. Software to analyze concentration is readily available, but software to analyze turnover is lacking. Data analysis workflows typically don’t access the full breadth of information about instrument precision and accuracy that is present in each peptide isotopic envelope measurement. This method utilizes both isotope distribution and changes in neutromer spacing, which benefits the analysis of both concentration and turnover.

Results

We have developed a data analysis tool, DeuteRater, to measure protein …

Annotation And Identification Of Several Glycerolipid Metabolic Related Ortholog Genes; Mrub_0437, Mrub_1813 And Mrub_2759 In The Organism Meithermus Ruber And Their Predicted Respective Orthologs B3926, B4042 And Bo514 Found In E.Coli., Abdul Rahman Abdul Kader, Dr. Lori R. Scott

Meiothermus ruber Genome Analysis Project

We predict Mrub_0437 encodes the enzyme glycerol kinase (DNA coordinates [417621..419183), which is an intermediary step of the glycerolipid metabolic pathway (KEGG map00561), It catalyzes the conversion of glycerol to sn-Glycerol-3-phosphate. The E. coli K12 MG1655 ortholog is predicted to be b3926.

We predict Mrub_1813 encodes the enzyme diacylglycerol kinase (DNA coordinates [1864659..1865063), which is an intermediary step of the glycerolipid metabolic pathway (KEGG map00561), It catalyzes the conversion of 1,2-diacyl-sn-glycerol to 1,2-diacyl-sn-glycerol 3-phosphate. The E. coli K12 MG1655 ortholog is predicted to be b4042.

We predict Mrub_2759 encodes the enzyme glycerol kinase (DNA coordinates [2799712..2800665), which is an intermediary …

Transposon-Mediated Stable Suppression Of Gene Expression In The Developing Chick Retina, Masaru Nakamoto, Chizu Nakamoto

Biology Faculty Publications

The embryonic chick has long been a favorite model system for in vivo studies of vertebrate development. However, a major technical limitation of the chick embryo has been the lack of efficient loss-of-function approaches for analyses of gene functions. Here, we describe a methodology in which a transgene encoding artificial microRNA sequences is introduced into embryonic chick retinal cells by in ovo electroporation and integrated into the genome using the Tol2 transposon system. We show that this methodology can induce potent and stable suppression of gene expression. This technique therefore provides a rapid and robust loss-of-function approach for studies of …

The Assembly Factor Pet117 Couples Heme A Synthase Activity To Cytochrome Oxidase Assembly, Nicholas G. Taylor, Samantha Swenson, Nicholas J. Harris, Edward M. Germany, Jennifer L. Fox, Oleh Khalimonchuk

Department of Biochemistry: Faculty Publications

Heme a is an essential metalloporphyrin cofactor of the mitochondrial respiratory enzyme cytochrome c oxidase (CcO). Its synthesis from heme b requires several enzymes, including the evolutionarily conserved heme a synthase (Cox15). Oligomerization of Cox15 appears to be important for the process of heme a biosynthesis and transfer to maturing CcO. However, the details of this process remain elusive, and the roles of any additional CcO assembly factors that may be involved remain unclear. Here we report the systematic analysis of one such uncharacterized assembly factor, Pet117, and demonstrate in Saccharomyces cerevisiae that this evolutionarily conserved protein is necessary for …

Structural And Mechanistic Insights Into Hemoglobincatalyzed Hydrogen Sulfide Oxidation And The Fate Of Polysulfide Products, Victor Vitvitsky, Pramod K. Yadav, Sojin An, Javier Seravalli, Uhn-Soo Cho, Ruma V. Banerjee

Department of Biochemistry: Faculty Publications

Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize H₂S but, lacking organelles, cannot dispose of H₂S via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes H₂S to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates. Here, we report the crystal structure of human hemoglobin containing low spin ferric sulfide, the first intermediate in heme-catalyzed sulfide oxidation. The structure provides molecular insights into why sulfide is susceptible to oxidation in human …

Short Carboxylic Acid-Carboxylate Hydrogen Bonds Can Have Fully Localized Protons, Jiusheng Lin, Edwin Pozharski, Mark A. Wilson

Department of Biochemistry: Faculty Publications

Short hydrogen bonds (H-bonds) have been proposed to play key functional roles in several proteins. The location of the proton in short H-bonds is of central importance, as proton delocalization is a defining feature of low barrier hydrogen bonds (LBHBs). Experimentally determining proton location in H-bonds is challenging. Here, bond length analysis of atomic (1.15–0.98 Å) resolution X-ray crystal structures of the human protein DJ-1 and its bacterial homolog YajL was used to determine the protonation states of H-bonded carboxylic acids. DJ-1 contains a buried, dimer-spanning 2.49 Å H-bond between Glu15 and Asp23 that satisfies standard donor-acceptor distance criteria for …

End-To-End Molecular Communication Channels In Cell Metabolism: An Information Theoretic Study, Zahmeeth Sayed Sakkaff, Jennie L. Catlett, Mikaela Cashman, Massimiliano Pierobon, Nicole R. Buan, Myra B. Cohen, Christine A. Kelley

Department of Biochemistry: Faculty Publications

The opportunity to control and fine-tune the behavior of biological cells is a fascinating possibility for many diverse disciplines, ranging from medicine and ecology, to chemical industry and space exploration. While synthetic biology is providing novel tools to reprogram cell behavior from their genetic code, many challenges need to be solved before it can become a true engineering discipline, such as reliability, safety assurance, reproducibility and stability. This paper aims to understand the limits in the controllability of the behavior of a natural (non-engineered) biological cell. In particular, the focus is on cell metabolism, and its natural regulation mechanisms, and …

Portulaca Oleracea Extract Can Inhibit Nodule Formation Of Colon Cancer Stem Cells By Regulating Gene Expression Of Notch Signal Transduction Pathway, Li Chen

Dissertations, Master's Theses and Master's Reports

The main aim of this study was to investigate the effects of Portulaca oleracea extract on tumor formation in colon cancer stem cells and chemotherapy sensitivity. In addition, this study analyzed the genetic changes within the Notch signal transduction pathway associated with the effects of the extracts. Serum-free cultures of colon cancer cells (HT-29) and HT-29 stem cells were treated with the chemotherapeutic drug 5-FU to assess sensitivity. Injections of the stem cells were also given to BALB/c mice to confirm tumor growth and characteristics. In addition, the effect of different concentrations of P. oleracea extract was tested on the …

Hyper-Activation Of Pp60(Src) Limits Nitric Oxide Signaling By Increasing Asymmetric Dimethylarginine Levels During Acute Lung Injury, Sanjiv Kumar, Xutong Sun, Satish Kumar Noonepalle, Qing Lu, Evgeny Zemskov, Ting Wang, Saurabh Aggarwal, Christine Gross, Shruti Sharma, Ankit A. Sesai, John D. Catravas

Bioelectrics Publications

The molecular mechanisms by which the endothelial barrier becomes compromised during lipopolysaccharide (LPS) mediated acute lung injury (ALI) are still unresolved. We have previously reported that the disruption of the endothelial barrier is due, at least in part, to the uncoupling of endothelial nitric oxide synthase (eNOS) and increased peroxynitrite-mediated nitration of RhoA. The purpose of this study was to elucidate the molecular mechanisms by which LPS induces eNOS uncoupling during ALI. Exposure of pulmonary endothelial cells (PAEC) to LPS increased pp60^Src activity and this correlated with an increase in nitric oxide (NO) production, but also an increase in …

The Role Of Alternative Polyadenylation Mediated By Cpsf30 In Arabidopsis Thaliana, Guijie Hao

Theses and Dissertations--Plant and Soil Sciences

Drought stress is considered one of the most devastating abiotic stress factors that limit crop productivity for modern agriculture worldwide. There is a large range of physiological and biochemical responses induced by drought stress. The responses range from physiological and biochemical to regulation at transcription and posttranscriptional levels. Post-transcription, the products encoded by eukaryotic genes must undergo a series of modifications to become a mature mRNA. Polyadenylation is an important one in terms of regulation. Polyadenylation impacts gene expression through determining the coding and regulation potential of the mRNA, especially when different mRNAs from the same gene may be polyadenylated …

Analysis Of Microbial Diversity In Disturbed Soil, Tyler G. Sanda

Williams Honors College, Honors Research Projects

This paper uses the composition and abundance of microbial species to analyze soil recovery in disturbed land. Surface mining disturbs ecological communities throughout the world. As organizations seek to reclaim these disturbed lands, a proper analysis of recovery is needed. In previous studies, recovery of disturbed land was limited to surface examinations, which do not characterize the possible unseen devastating effects of the subsoil. Soil microorganisms are extremely sensitive to environmental changes such as strip mining. It is proposed that these microorganisms may serve as better indicators of recovery post disturbance. Our analysis indicates microbial recovery, however it may not …

Significant Enhancement Of Fatty Acid Composition In Seeds Of The Allohexaploid, Camelina Sativa, Using Crispr/Cas9 Gene Editing, Wen Zhi Jhang, Isabelle M. Henry, Peter G. Lynagh, Lucia Comai, Edgar B. Cahoon, Donald P. Weeks

Department of Biochemistry: Faculty Publications

The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were associated with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e. a decrease from ~16% to <4%) and linolenic acid (a decrease from ~35% to <10%). These changes result in oils that are superior on multiple levels: they are healthier, more oxidatively stable and better suited for production of certain commercial chemicals, including biofuels. As expected, A. …