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Aminoacyl-Trna Synthetase Complexes: Molecular Multitasking Revealed, Corinne D. Hausmann, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

The accurate synthesis of proteins, dictated by the corresponding nucleotide sequence encoded in mRNA, is essential for cell growth and survival. Central to this process are the aminoacyl-tRNA synthetases (aaRSs), which provide amino acid substrates for the growing polypeptide chain in the form of aminoacyl-tRNAs. The aaRSs are essential for coupling the correct amino acid and tRNA molecules, but are also known to associate in higher order complexes with proteins involved in processes beyond translation. Multiprotein complexes containing aaRSs are found in all three domains of life playing roles in splicing, apoptosis, viral assembly, and regulation of transcription and translation. …

Electron Crystallography Of Aquaporins, Simeon Andrews, Steve Reichow, Tamir Gonen

Chemistry Faculty Publications and Presentations

Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. We describe features of aquaporins common to both electron and X-ray crystallographic structures; as well as some structural insights unique to electron crystallography, including aquaporin junction formation and lipid-protein interactions.

Sphingolipid Long-Chain Base Hydroxylation Is Important For Growth And Regulation Of Sphingolipid Content And Composition In Arabidopsis, Ming Chen, Jennifer E. Markham, Charles R. Dietrich, Jan G. Jaworski, Edgar B. Cahoon

Department of Biochemistry: Faculty Publications

Sphingolipids are structural components of endomembranes and function through their metabolites as bioactive regulators of cellular processes such as programmed cell death. A characteristic feature of plant sphingolipids is their high content of trihydroxy long-chain bases (LCBs) that are produced by the LCB C-4 hydroxylase. To determine the functional significance of trihydroxy LCBs in plants, T-DNA double mutants and RNA interference suppression lines were generated for the two Arabidopsis thaliana LCB C-4 hydroxylase genes Sphingoid Base Hydroxylase1 (SBH1) and SBH2. These plants displayed reductions in growth that were dependent on the content of trihydroxy LCBs in sphingolipids. …

Promoter Library Designed For Fine-Tuned Gene Expression In Pichia Pastoris, Franz S. Hartner, Claudia Ruth, David Langenegger, Sabrina N. Johnson, Petr Hyka, Geoff P. Lin-Cereghino, Joan Lin-Cereghino, Karin Kovar, James Cregg, Anton Glieder

College of the Pacific Faculty Articles

Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (P_AOX1) sequence. This first library initially spanned an activity range between ∼6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing …

Pge2 Receptor Subsets Mediate Hcg Regulation Of The Plasminogen Activator/Plasmin System In Primate Periovulatory Follicles, Nune Markosyan

Theses and Dissertations in Biomedical Sciences

Elevated follicular prostaglandin E2 (PGE2) levels are necessary for follicle rupture during ovulation. As the degradation of the follicle wall requires proteolysis, it was hypothesized that regulation of proteolysis by gonadotropins is PGE2 mediated.

There are four PGE2 receptors: EP1, EP2, EP3 and EP4. To determine which PGE2 receptors are present in monkey periovulatory follicles, granulosa cells and whole ovaries were collected 0, 12, 24, and 36 hours after administration of an ovulatory dose of human chorionic gonadotropin (hCG), or 36 hours after hCG from monkeys that received the prostaglandin (PG) synthesis inhibitor celecoxib. All EP receptor mRNAs were present …

Nanosecond Pulsed Electric Fields Induce A Mitochondria-Independent Apoptosis In B16f10 Melanoma Cells In Vitro, Wentia Elissa Ford

Theses and Dissertations in Biomedical Sciences

Nanosecond pulsed electric fields (nsPEFs) are ultra-short pulses that induce direct electric field and biological effects that initiate apoptosis. Here the application of ten 300ns pulses ranging in electric fields from 12kV/cm-60kV/cm was administered to determine the effects on B16F10 melanoma cells evaluated by in vitro studies. Initial application of nsPEFs demonstrated apoptosis induction in an electric field- and pulse number-dependent manner measured by caspase activation that correlated with decrease in cell viability 24hr post pulse. In addition caspase activity was shown to be independent of calcium mobilization though ions may play a part in other aspects of apoptosis. The …

In Vivo Murine Melanoma Tumor Responses To Nanosecond Pulsed Electric Field Treatment, Xinhua Chen

Theses and Dissertations in Biomedical Sciences

High intensity nanosecond pulsed electric fields (nsPEF) were applied to melanoma tumors to observe functional and structural biological changes and to investigate the possible molecular mechanisms responsible. An animal model was set up by injecting B16F10 mouse melanoma cells into SKH-1 mice. A treatment (Tx) of 100 pulses: 300 nanosecond duration; 40 kV/cm field strength; at 0.5 Hz rate were delivered to melanoma tumors in 120 mice. The nsPEF Txcaused tumor self-destruction with sharply decreased cell volumes and shrunken nuclei. The apoptotic biochemical tests confirmed nsPEF Tx induced apoptosis in a time-dependent manner. Examination of gross vessel and micro-vessel density …

Evolution Of Metal(Loid) Binding Sites In Transcriptional Regulators, Efrén Ordóñez, Saravanamuthu Thiyagarajan, Jeremy D. Cook, Timothy L. Stemmler, José A. Gil., Luís M. Mateos, Barry P. Rosen

Biochemistry and Molecular Biology Faculty Publications

Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II) and Ni(II). These homodimeric repressors bind to DNA in absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, and the …

The Role Of Upstream Sequences In Selecting The Reading Frame On Tmrna, Allen R. Buskirk, Mickey R. Miller, David W. Healey, Jonathan D. Dewey, Stephen G. Robison

Faculty Publications

tmRNA acts first as a tRNA and then as an mRNA to rescue stalled ribosomes in eubacteria. Two unanswered questions about tmRNA function remain: how does tmRNA, lacking an anticodon, bypass the decoding machinery and enter the ribosome? Secondly, how does the ribosome choose the proper codon to resume translation on tmRNA? According to the -1 triplet hypothesis, the answer to both questions lies in the unique properties of the three nucleotides upstream of the first tmRNA codon. These nucleotides assume an A-form conformation that mimics the codon-anticodon interaction, leading to recognition by the decoding center and choice of the …

Platyhelminth Mitochondrial And Cytosolic Redox Homeostasis Is Controlled By A Single Thioredoxin Glutathione Reductase And Dependent On Selenium And Glutathione, Mariana Bonilla, Ana Denicola, Sergey V. Novoselov, Anton A. Turanov, Anna Protasio, Darwin Izmendi, Vadim N. Gladyshev, Gustavo Salinas

Vadim Gladyshev Publications

Platyhelminth parasites are a major health problem in developing countries. In contrast to their mammalian hosts, platyhelminth thiol-disulfide redox homeostasis relies on linked thioredoxin-glutathione systems, which are fully dependent on thioredoxin-glutathione reductase (TGR), a promising drug target. TGR is a homodimeric enzyme comprising a glutaredoxin domain and thioredoxin reductase (TR) domains with a C-terminal redox center containing selenocysteine (Sec). In this study, we demonstrate the existence of functional linked thioredoxin-glutathione systems in the cytosolic and mitochondrial compartments of Echinococcus granulosus, the platyhelminth responsible for hydatid disease. The glutathione reductase (GR) activity of TGR exhibited hysteretic behavior regulated by the …

The Response Of Hn4 Cells To Porphyromonas Gingivalis Dna, Cheyanne Warren

Theses and Dissertations

Periodontal disease is one of the most common human diseases. Bacteria trigger the onset and progression of the disease and among them Porphyromonas gingivalis has been demonstrated to be a major etiologic agent. Although the interaction of the bacterium with the host is of major importance for the understanding of the disease mechanisms, both the host as well as the pathogen components involved in the interaction remain poorly understood. One of the bacterial components capable of eliciting a host response is unmethylated CpG DNA motifs found in bacteria. Thus, the first aim was to determine the response of oral epithelial …

Critical Involvement Of The Atm-Dependent Dna Damage Response In The Apoptotic Demise Of Hiv-1-Elicited Syncytia, Jean-Luc Perfettini, Roberta Nardacci, Mehdi Bourouba, Frederic Subra, Laurent Gros, Claire Séror, Gwenola Manic, Filippo Rosselli, Alessandra Amendola, Peggy Masdehors, Luciana Chessa, Giuseppe Novelli, David M. Ojcius, Jan Konrad Siwicki, Magdalena Chechlinska, Christian Auclair, Jose R. Regueiro, Hughes De Thé, Marie-Lise Gougeon, Mauro Piacentini, Guido Kroemer

All Dugoni School of Dentistry Faculty Articles

DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM), which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown …

Structural And Functional Mapping Of The Archaeal Multi-Aminoacyl-Trna Synthetase Complex, Corinne D. Hausmann, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Methanothermobacter thermautotrophicus contains a multi-aminoacyl-tRNA synthetase complex (MSC) of LysRS, LeuRS and ProRS. Elongation factor (EF) 1A also associates to the MSC, with LeuRS possibly acting as a core protein. Analysis of the MSC revealed that LysRS and ProRS specifically interact with the idiosyncratic N- and C- termini of LeuRS, respectively. EF-1A instead interacts with the inserted CP1 proofreading domain, consistent with models for post-transfer editing by class I synthetases such as LeuRS. Together with previous genetic data, these findings show that LeuRS plays a central role in mediating interactions within the archaeal MSC by acting as a core scaffolding …

A Cytosolic Iron Chaperone That Delivers Iron To Ferritin, Haifeng Shi, Krisztina Z. Bencze, Timothy L. Stemmler, Caroline C. Philpott

Biochemistry and Molecular Biology Faculty Publications

Ferritins are the main iron storage proteins found in animals, plants and bacteria. The capacity to store iron in ferritin is essential for life in mammals, but the mechanism by which cytosolic iron is delivered to ferritin is unknown. Human ferritins expressed in yeast contain little iron. The human Poly r(C)-Binding Protein 1 (PCBP1) increased the amount of iron loaded into ferritin when expressed in yeast. PCBP1 bound to ferritin in vivo, and bound iron and facilitated iron loading into ferritin in vitro. Depletion of PCBP1 in human cells inhibited ferritin iron loading and increased cytosolic iron pools. Thus, PCBP1 …

Atf4 Is An Oxidative Stress–Inducible, Prodeath Transcription Factor In Neurons In Vitro And In Vivo, Philipp Lange, Juan Chavez, John T. Pinto, Giovanni Coppola, Chiao-Wang Sun, Tim Townes, Rajiv Ratan

NYMC Faculty Publications

Oxidative stress is pathogenic in neurological diseases, including stroke. The identity of oxidative stress-inducible transcription factors and their role in propagating the death cascade are not well known. In an in vitro model of oxidative stress, the expression of the bZip transcription factor activating transcription factor 4 (ATF4) was induced by glutathione depletion and localized to the promoter of a putative death gene in neurons. Germline deletion of ATF4 resulted in a profound reduction in oxidative stress-induced gene expression and resistance to oxidative death. In neurons, ATF4 modulates an early, upstream event in the death pathway, as resistance to oxidative …

Structural And Biochemical Investigation Of The Molecular Mechanisms Of Dna Response And Repair In Humans And Escherichia Coli., Steven Michael Shell

Electronic Theses and Dissertations

The genomes of all living cells are under constant attack from both endogenous and exogenous agents that damage DNA. In order to maintain genetic integrity a variety of response pathways have evolved to recognize and eliminate DNA damage. Replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, is a required factor for all major DNA metabolisms. Although much work has been done to elucidate the nature of the interaction between RPA and ssDNA currently there is no structural information on how the full-length protein binds to ssDNA. This study presents a novel examination of the full nucleoprotein complex …

Tendinopathy Discrimination By Use Of Spatial Frequency Parameters In Ultrasound B-Mode Images, Gregory R. Bashford, Nicholas Tomsen, Shruti Arya, Judith M. Burnfield, Kornelia Kulig

Biomedical Imaging and Biosignal Analysis Laboratory

The structural characteristics of a healthy tendon are related to the anisotropic speckle patterns observed in ultrasonic images. This speckle orientation is disrupted upon damage to the tendon structure as observed in patients with tendinopathy. Quantification of the structural appearance of tendon shows promise in creating a tool for diagnosing, prognosing, or measuring changes in tendon organization over time. The current work describes a first step taken towards this goal—classification of Achilles tendon images into tendinopathy and control categories. Eight spatial frequency parameters were extracted from regions of interest on tendon images, filtered and classified using linear discriminant analysis. Resulting …

Collagen 11a1 Is Indirectly Activated By Lymphocyte Enhancer-Binding Factor 1 (Lef1) And Negatively Regulates Osteoblast Maturation, Rachel A. Kahler, Sorcha M.C. Yingst, Luke H. Hoeppner, Eric D. Jensen, David Krawczak, Julia T. Oxford, Jennifer J. Westendorf

Biomedical Research Institute Publications and Presentations

Alpha 1 (XI) collagen (Col11a1) is essential for normal skeletal development. Mutations in Col11a1 cause Marshall and Stickler syndromes, characterized by craniofacial abnormalities, nearsightedness and hearing abnormalities. Despite its link to human diseases, few studies have characterized the factors that control Col11a1 transcription. We previously identified Col11a1 as a differentially expressed gene in Lef1-suppressed MC3T3 preosteoblasts. Here we report that Lef1 activates the Col11a1 promoter. This activation is dependent upon the DNA binding domain of Lef1, but does not require the ß-catenin interaction domain, suggesting that it is not responsive to Wnt signals. Targeted deletion of Col11a1 with an antisense …

Optimization Of Expression And Purification Of Acetoacetyl-Coa Thiolase From Sunflower, Anthony Muriithi Maina

Theses, Dissertations and Culminating Projects

The [beta]-oxidation system in sunflower (Helianthus annuus L.) cotyledons is distinguished by the existence of two different thiolase isoforms, Thiolase I (acetoacetyl-CoA thiolase, EC 2.3.1.9 AACT) and Thiolase II (3-oxoacyl-CoA thiolase, EC 2.3.1.16 OACT). Glyoxysomal AACT is the last enzyme in the p-oxidation of fatty acids in plant glyoxysomes. Glyoxysomal AACT has been successfully cloned, expressed and purified from sunflower cotyledons (Dyer et al., 2006). In this paper we investigate the optimal conditions for both expression and purification. These include the choice of vector, the growth conditions and the purification parameters for the maximum possible yield of AACT protein.

Influence Of Carbonate On The Binding Of Carboplatin To Dna, Robert S. Sorokanich

Renée Crown University Honors Thesis Projects - All

The reaction of aged carboplatin (reaction of carboplatin in 24 mM NaHCO₃ for 45 h, 37^o C, pH 8.6) with pBR322 DNA for 0 ≤ r ≤ 2.8, where r = [drug]/[DNA-bp], in 24 mM HEPES buffer, pH 7.4, for 24 h followed by agarose gel electrophoresis showed DNA mobility changes consistent with unwinding closed circular DNA. However, identical experiments conducted in a two buffer system, 24mM HEPES plus 24mM carbonate, showed no DNA mobility changes, indicating that carbonate blocks formation of the 1,2-intrastrand crosslink on DNA. Studies with aged carboplatin and with cisplatin carried out with 2.0 …

Expressing A Mammalian Signaling Protein In E. Coli, Soyika A. Richardson

Renée Crown University Honors Thesis Projects - All

Much of what is currently known about GPCR structure was based on X-ray crystallographic measurements of rhodopsin. Furthermore, due to its inexpensiveness and the availability of simple methods to purify it in large quantities from cow eyes, bovine rhodopsin has been successfully used in obtaining diffraction-quality three-dimensional crystals. In our experiment, the bovine rhodopsin protein was cloned into the commercially available E. coli expression plasmid, pBAD TOPO^®, using forward and reverse PCR primers designed in our lab. Plasmid DNA from these cells was purified in order to analyze the direction of the inserted gene. The purified plasmid DNA …

Hypochlorous Acid Mediated Damage Of Chromatin, Joseph Insugn Kang

Loma Linda University Electronic Theses, Dissertations & Projects

Hypochlorous acid (HOCl), the primary product of activated neutrophils at sites of inflammation, can damage both DNA and associated histone proteins. HOCl damage to cytosine bases in DNA generates the chlorination damage products 5-chlorocytosine (ClC) and 5-chlorouracil (ClU) as well as the oxidation damage products 5- hydroxycytosine (HOC) and 5-hydroxyuracil (HOU). Histone damage by HOCl can be measured by quantitation of 3-chlorotyrosine and 3,5-dichlorotyrosine, stable and unique markers of protein damage caused by HOCl. The studies presented here investigate how and where these damage products occur and what implications they might have in the correlation between chronic inflammation and carcinogenesis. …

Detritus Type Alters The Outcome Of Interspecific Competition Between Aedes Aegypti And Aedes Albopictus (Diptera : Culicidae), Ebony G. Murrell, Steven A. Juliano

Faculty Publications – Biological Sciences

Many studies of interspecific competition between Aedes albopictus (Skuse) and Aedes aegypti (L.) (Diptera: Culicidae) larvae show that Ae. albopictus are superior resource competitors to Ae. aegypti. Single-species studies indicate that growth and survival of Ae. albopictus and Ae. aegypti larvae are affected by the type of detritus present in containers, which presumably affects the amount and quality of microorganisms that the mosquito larvae consume. We tested whether different detritus types alter the intensity of larval competition by raising 10 different density/species combinations of Ae. albopictus and Ae. aegypti larvae under standard laboratory conditions, with one of four detritus types …

Lipid Aldehydes Are Substrates And Transcriptional Regulators Of Cytochromes P450., Immaculate Amunom

Electronic Theses and Dissertations

4-hydroxy-2-nonenal (4-HNE) and propene-2-al (acrolein) are highly reactive á,â-unsaturated aldehydes. 4-HNE and acrolein are generated in vivo as products of lipid peroxidation. These aldehydes are implicated in the onset of several diseases including atherosclerosis and neurodegenerative disease. They also react with nucleophilic cellular macromolecules including proteins, DNA and phospholipids. Some of these reactions in vivo lead to inhibition of enzyme activities, depletion of glutathione and oxidative stress. Because of their pathophysiological relevance, attention has focused during the last decade on the ability of mammals to metabolize these lipid aldehydes. The purpose of this study was therefore to determine the role …

Identification Of Metallo-Ss-Lactamase Inhibitors Via Enzyme Assay And Hplc-Ms Monitoring, Peggy Ann Sanchez

Theses, Dissertations and Culminating Projects

The vast overuse of antibiotics along with extrachromosomal gene insertion between different bacterial species has led to the Darwinian consequence of antibiotic resistance. Amongst the contributing factors to the rise of bacterial resistances are the production of ß-lactamases, which pose a major impediment to treatment with ß-lactam drugs such as penicillins and carbapenems. SPM-1 is a metallo-ß-lactamase (MBL) that confers antibiotic resistance to bacteria through zinc-mediated hydrolysis of ß-lactams. Bacteria that express the SPM-1 metalloenzyme show reduced sensitivity to ß-lactam drugs. One method to defeat this form of resistance is to administer a ß-lactam antibiotic with a potent ß-lactamase inhibitor …

Expression Analysis Of Plant Defense Responses During The Establishment Of Biotrophy And Role Of Abiotic Stress In The Infection Of Dyer’S Woad (Isatis Tinctoria) By Puccinia Thlaspeos, Elizabeth Thomas

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The kinetics and amplitude of the salicylic acid-responsive pathogenesis-related (PR) genes and the cytochrome P450 gene ItCYP79B2 in the compatible interaction between Puccinia thlaspeos and dyer’s woad (Isatis tinctoria) during the first 72 hours of inoculation were examined. Immediately following penetration of the host by the rust pathogen, there was a modest up-regulation of PR genes but a significant down-regulation of ItCYP79B2 expression. During haustoria formation, a significant pathogen-mediated suppression of PR genes was observed with a corresponding up-regulation of ItCYP79B2. This potentially facilitates haustoria formation by P. thlaspeos. After haustoria formation, a more …

Intracellular Levels Of Centrin2, Xpc, & Hhr23b Increase After Dna Damage, Nicholas A. Rowland

Honors Capstones

Capstone submitted as a graduate requirement for the BSU Honors Program.

Cloning And Biochemical Characterization Of The Recq Helicase And Topoisomerase Iii In Schizosaccharomyces Pombe, Rebecca Wood

All Theses And Dissertations

RecQ and Topoisomerase III proteins are both essential for proper chromosomal maintenance in species ranging from bacteria to humans. Due to their highly conserved nature throughout evolution, understanding their basic biochemical properties as well as their interactions together is important for understanding human health, disease, and even to provide potential evolutionary insight. S. pombe has been chosen as a model organism because little research has been done on the biochemical analysis of its RecQ homolog, Rqhl. This study shows developments in the biochemical characterization of a truncation protein of Rqh1 including AFM (Atomic Force Microscopy) Imaging, DNA binding, and ATP …

Dioxidobis(2-Oxo-1,2-Dihydropyridin-3-Olato)­Molybdenum(Vi), Manoj Trivedi, Daya Pandey, Nigam Rath

Chemistry & Biochemistry Faculty Works

In the title compound, [Mo(C5H4NO2)2O2], the MoVI atom exhibits a distorted octa­hedral coordination geometry formed by two terminal oxo ligands and two monoanionic O,O-bidentate pyridinone ligands. The two terminal oxo ligands lie in a cis arrangement, the ketonic O atoms of the pyridinone ligands are coordinated trans to the oxo ligands and the deprotonated hydroxyl O atoms are located trans to each other. The crystal structure contains inter­molecular N-H...O hydrogen bonds, C-H...O contacts and face-to-face [pi]-[pi] stacking inter­actions with an inter­planar separation of 3.25 (1) Å.

Diethyl 2-[(4-Nitro­Phen­Yl)(4-Phenyl-1,2,3-Selenadiazol-5-Yl)Meth­Yl]Malonate, A. Marx, S. Saravanan, S. Muthusubramanian, V. Manivannan, Nigam Rath

Chemistry & Biochemistry Faculty Works

In the title compound, C22H21N3O6Se, the heterocyclic ring makes dihedral angles of 50.03 (11) and 67.75 (11)°, respectively, with the benzene and phenyl rings. The terminal C atoms of the ester groups are disordered over two positions: the site occupancies for the C atoms are 0.62 (3)/0.38 (3) and 0.48 (3)/0.52 (3). In the crystal structure, weak intra- and inter­molecular C-H...O inter­actions are observed.