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Biochemistry and Molecular Biology

Permanent URI for this collectionhttp://hdl.handle.net/10342/77

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  • ItemEmbargo
    Defining the Metabolic and Regulatory Properties of the mdo Operon in Pseudomonas aeruginosa PAO1
    (East Carolina University, July 2024) Adindu, Chukwuemeka Steve
    Pseudomonas aeruginosa PAO1 is an opportunistic human pathogen that is especially problematic for individuals with cystic fibrosis and immunocompromised patients. It is a leading cause of nosocomial infections and is responsible for 10% of all hospital-acquired infections. The CDC classifies the organism as a serious threat mainly due to its emerging development of multidrug resistance. Several virulence factors contribute to P. aeruginosa PAO1 pathogenicity including hydrogen sulfide. Sulfide (HS-) at sub-micromolar concentrations protects P. aeruginosa PAO1 from antibiotic-induced oxidative damage and host-produced reactive oxygen species. However, elevated sulfide levels result in cellular toxicity and affect the organism’s ability to form a biofilm. Therefore, HS- concentrations must be tightly regulated to balance the potential toxicity with bacterial virulence. In several organisms, toxic levels of HS- are converted to usable sulfur forms by the combined actions of dioxygenases and sulfurtransferases. Increased levels of HS- results in the formation of low molecular weight persulfides which are the substrates for the sulfide oxidation enzymes. In P. aeruginosa PAO1, enzymes expressed on the mdo operon have been identified but their mechanism of action and metabolic roles have not been elucidated. In P. aeruginosa PAO1, the mdo operon expresses 3-mercaptopropionate dioxygenase (MDO) and a sulfurtransferase (ST). MDO was initially characterized as utilizing 3-mercaptopropionate (3MPA) as a substrate; however, P. aeruginosa PAO1 could not utilize 3MPA in microbial growth studies to ascertain the physiological relevance of the thiol substrate. MDO was able to oxidize 3-mercaptopyruvate (3MPR), which is a physiologically relevant substrate and can also be linked with ST activity. Even though MDO is expressed from the same operon as an annotated ST, the functional role of the ST enzyme has not been recognized. ST enzymes have conserved cysteine residues that mediate sulfur transfer from a sulfur donor to a sulfur acceptor. These sulfur donors and acceptors are usually low molecular weight thiols that are ubiquitous in cells. The ST expressed on the mdo operon has four rhodanese domains with two of the domains containing putative catalytic Cys residues (Cys191 and Cys435) with the potential to mediate sulfur transfer through an enzyme cysteine persulfide intermediate. Cys435 was the only accessible thiol in thiol assays. Results from HDX-MS investigations supported a more solvent-accessible region surrounding Cys435. The accessible thiol was identified as Cys435 in HDX-MS investigations, which corresponded to the role of this residue as the sulfide mediator. Only Cys435 formed a persulfide intermediate with either thiosulfate or 3MPR as the sulfur donor in cysteine persulfidation assays. These studies support Cys435 as the catalytic cysteine with thiosulfate and 3MPR serving as the sulfur donor. ST showed a similar affinity for 3MPR and 3MPA suggesting that each substrate could serve as a putative sulfur acceptor for the enzyme to form a persulfide. MDO was able to oxidize the persulfides of 3MPA and 3MPR, suggesting a metabolic link between MDO and ST. Although 3MPA could bind ST and serve as a substrate for MDO, it was not a viable sulfur source in growth studies. Proteomic studies were performed to identify the changes in protein expression when the organism was grown in sulfur-free media supplemented with sulfide. When P. aeruginosa PAO1 is under sulfide stress, the genes that were high in abundance were those involved in biological processes including amino acid breakdown, arginine deaminase pathway, and tRNA metabolism amongst other pathways. The data from these investigations point to the potential of the enzymes of the mdo operon to mobilize and assimilate sulfide in P. aeruginosa PAO1 thereby enhancing its viability and pathogenicity. Understanding the strategies for the acquisition of sulfur from less preferred sources and their regulation provides insights into the metabolic versatility and pathogenicity of P. aeruginosa PAO1, highlighting potential targets for therapeutic interventions.
  • ItemOpen Access
    An NTP-Driven Mechanism for the Nucleotide Addition Cycle of Escherichia Coli RNA Polymerase During Transcription
    (2022-10-25) Johnson, Ronald S.; Strausbauch, Mark; McCloud, Christopher
  • ItemOpen Access
    HUMAN RNA METHYLTRANSFERASE METTL16 AFFECTS MULTIPLE CELL PROCESSES THROUGH DIFFERENTIAL FUNCTION OF ITS DOMAINS
    (East Carolina University, 2022-11-10) Talic, Emily Satterwhite
    ABSTRACT In recent years, disease therapies have been developed to target specific RNA and protein expression changes in the affected cells/tissue. The RNA post-transcriptional modification, methyl-6-adenosine (m6A), has been implicated in a number of disease-states as a driver of aberrant RNA and protein expression. m6A is the reversible methylation of adenosine which is produced by enzymes known as m6A RNA methyltransferases. There are currently two known m6A RNA methyltransferases which methylate messenger RNA in humans: METTL3/14 complex, and METTL16. The METTL3/14 complex has been widely implicated and studied in cancer development and progression, while much of METTL16 has not been investigated. While a few studies have probed the biochemical details of METTL16, cancer studies have shown METTL16 protein expression level changes have been associated with a variety of cancer types. The research in this dissertation project was performed in an effort to determine the RNAs affected by and the cell-wide effects of METTL16’s function. Using RNA immunoprecipitation, the studies here show that METTL16 binds a number of RNAs outside the 3 that are currently accepted as RNA targets. It was also discovered that METTL16 is localized to both the nucleus and cytoplasm, suggesting a role in addition to methylation in the cell. Mutation of key residues in METTL16 protein resulted in a large number of changes in RNA and protein expression levels determined with RNA sequencing, real-time PCR, and mass spectrometry proteomics. A majority of these RNAs and proteins are associated with cytoskeletal maintenance, calcium signaling, and cell cycle regulation, all of which have implications in distinct diseases including cancer. Of interest, only some of the METTL16 mutations seemed to affect cell cycle phase occupancy. Given the multiple changes seen among cells containing mutated METTL16, it is plausible that this protein could aid in presentation or progression of associated diseases and could therefore be a justified target for cell therapies.
  • ItemOpen Access
    Maternal Aerobic Exercise, but Not Blood Docosahexaenoic Acid and Eicosapentaenoic Acid Concentrations, during Pregnancy Influence Infant Body Composition
    (2022-07-07) Kew, Kimberly A.; Houmard, Joseph A.; Tulis, David A.; Pawlak, Roman; Newton, Edward; Isler, Christy; DeVente, James; May, Linda E.
  • ItemOpen Access
    The Influence of Maternal Aerobic Exercise, Blood DHA and EPA Concentrations on Maternal Lipid Profiles
    (2022-03-16) Kew, Kimberly A.; Houmard, Joseph A.; Tulis, David A.; Pawlak, Roman; Newton, Edward; May, Linda E.
  • ItemOpen Access
    Functional Analysis of N-acetylglucosaminyltransferase-I Knockdown in 2D and 3D Neuroblastoma Cell Culturess
    (2021-11-11) Hall, M. Kristen; Burch, Adam P.; Schwalbe, Ruth A.
  • ItemOpen Access
    Caldesmon Ablation in Mice Causes Umbilical Herniation and Alters Contractility of Fetal Urinary Bladder Smooth Muscle
    (2021-04-21) Putz, Sandra; Barthel, Lisa Sophie; Frohn, Marina; Metzler, Doris; Barham, Mohammed; Pryymachuk, Galyna; Trunschke, Oliver; Lubomirov, Lubomir T.; Hescheler, Jürgen; Chalovich, Joseph; Neiss, Wolfram F.; Koch, Manuel; Schroeter, Mechthild M.; Pfitzer, Gabriele
  • ItemOpen Access
    THE DISTINCT ROLES OF TWO eIF4E ISOFORMS AND THEIR COGNATE 4EIPS IN THE GERMLINE OF C. elegans
    (East Carolina University, 8/5/2020) Huggins, Hayden P
    Biological information becomes functional at the step of mRNA translation when the protein product is synthesized. Translational regulation of mRNAs is critically important for proper modulation of gene expression in germ cells, gametes, and embryos. The ability of the nucleus to control gene expression in these systems may be limited due to spatial or temporal constraints, as well as the breadth of gene products they express to prepare for the rapid development that follows. During development, germ granules are hubs of post-transcriptional regulation of mRNAs. They assemble and remodel messenger ribonucleoprotein (mRNP) complexes that control translational repression and activation. Recently, mRNPs have been appreciated as discrete regulatory units, whose function is dictated by the many positive and negative acting factors within the complex. Repressed mRNPs must be remodeled and activated for translation on ribosomes to introduce novel proteins into germ cells. The eIF4E:eIF4Einteracting protein (4EIP) node controls many aspects of mRNP fate including localization, stability, poly(A)-elongation, deadenylation, and translational activation/repression. Furthermore, plant and animal species have evolved to express multiple functionally distinct eIF4E and 4EIP variants within germ cells, giving rise to different modes of translational regulation. Here we investigate the physiological and translational functions of two distinct eIF4E isoforms, IFE-1 and IFE-3, and their cognate 4EIPs (PGL-1 and IFET-1 respectively) in the germline of the nematode C. elegans. We find that deficiencies in IFE-1 and IFE-3 result in unique terminal phenotypes, suggesting non-redundant functions of these two closely related proteins. Each isoform contributes to development by regulating the translation of a unique pool of mRNAs. We provide biochemical evidence that IFE-1 and IFE-3 reside in discrete mRNPs with their cognate 4EIPs, that have P granule and P body related functions respectively. Additionally, we find that these 4EIPs control the localization of their respective eIF4E and likely their differential translational functions.
  • ItemOpen Access
    NMR method for monitoring changes in the core of lipoprotein particles in metabolism and disease
    (2017-01-14) Cistola, David; Robinson, Michelle
    A method is disclosed for measuring the properties of protein and lipoprotein elements in a sample. The method includes the of placing a small volume of a sample into a NMR instrument tuned to measure a particular nucleus, applying a series of radiofrequency pulses with intermittent delays in order to measure spin-spin and/or spin-lattice relaxation time constants from the time-domain decay of the signal, without the use of chemical shifts and without converting data into the frequency domain by Fourier transform or other means, at least partially suppressing the water signal prior to the beginning of a sequence used to record relaxation time constants in the time domain, optionally utilizing relaxation contrast agents or other chemical additives to perturb the solvent water or other elements of the sample, analyzing the exponentially decaying NMR signal in the time domain using multi-exponential analysis, and comparing differences in the relaxation time constants for lipoprotein- or protein-specific elements within a single human subject, or between subjects, to assess normal and abnormal metabolism reflective of increased disease risk or active disease.
  • ItemOpen Access
    Disruption of pqs synthesis using precursor analogs
    (2004-01-08) Pesci, Everett; Coleman, James P.
    The present invention concerns methods of detecting bacterial infections as well as methods of treating such infections with compounds such as methyl anthranilate. The detection and treatment of Pseudomonas is particularly preferred.
  • ItemOpen Access
    Measles vaccine vectors and methods of use
    (2003-03-20) Ross, Ted; Robinson, Harriet
    The present invention provides a novel vaccine expressing a fusion of the measles hemagglutinin (H) protein and the complement component, C3d, to enhance the titers of neutralizing antibody. Plasmids were generated that expressed a secreted (s) form of H and the same form fused to three tandem copies of the murine homologue of C3d (sH-3C3d). Analysis of titers of the antibody raised in vaccinated mice indicated that groups vaccinated with the DNA construct expressing sH-3C3d had higher titers of anti-H antibodies compared to serum from mice vaccinated with DNA expressing sH only. In addition, sH-3C3d elicited higher neutralizing antibody titers that inhibited MV induced plaque formation.
  • ItemOpen Access
    Erythropoietin ameliorates chemotherapy-induced toxicity in vivo
    (2002-10-17) Sigounas, George; Mehlhop, Paul
    A method of reducing organ toxicity in a subject being administered a cytotoxic agent comprising concurrently administering with the cytotoxic agent erythropoietin (EPO), the EPO being administered in an amount effective to reduce pulmonary toxicity caused by the cytotoxic agent.
  • ItemOpen Access
    Composition and formulations and their use as nociceptic, anti-anxiolytic and anabolic agents
    (2000-07-13) Nyce, Jonathan W.
    Composition and formulations comprising a first agent such as folinic acid, pharmaceutically acceptable salts thereof or mixtures thereof, and a second agent(s) such as analgesics, muscle relaxants, mood disorder agents, anti-inflammatories, anti-migraine agents, anti-emetics, diuretics, high protein composites, and the like. The products are suitable as nociceptics and for the treatment of wasting disorders, bulimia, anorexia nervosa, anxiety, irritability and other symptoms associated with Pre Menstrual Syndrome, as well as for administration either in conjunction with steroids or to compensate adenosine depletion and/or bizarre behavior or aggression common in steroid users.
  • ItemOpen Access
    Catabolite repression control (crc) gene and pseudomonas virulence
    (2002-08-22) Phibbs, Paul; Collier, David N.; Hager, Paul
    A method of screening for compounds that inhibit the virulence of Pseudomonas bacteria comprises the steps of: providing a culture medium comprising Pseudomonas bacteria; administering a test compound to said bacteria; and then detecting the presence or absence of inhibition of the catabolite repression control (Crc) protein in said bacteria, the inhibition of the Crc protein indicating said compound has antivirulence activity against Pseudomonas bacteria. Antivirulence compounds and the use thereof in treating Pseudomonas infections are also disclosed.
  • ItemOpen Access
    Method for the synthesis of 3-substituted indolizine and benzoindolizine compounds
    (2006-07-13) Hayford, Anthony; Kaloko, Joseph
    A method of making a compound of Formula (I) comprises reacting a compound of Formula (II) with a compound such as R1OH or R1SH, to produce said compound of Formula (I). Compounds of Formula (I) are useful, among other things, as dyes, spectral sensitizers, glycosidase inhibitors, and as antibacterial, antiviral, and anti-inflammatory agents.
  • ItemOpen Access
    Novel autoinducer molecules and uses therefor
    (2002-03-07) Greenberg, Everett; Iglewski, Barbara; Kende, Andrew; Milbank, Jared B. J.; Pearson, James P.; Pesci, Everett
    Novel bacterial quinolone signal molecules and, more particularly, pseudomonas quinolone signal ('PQS') molecules, e.g., 2-heptyl-3hydroxy-4-quinolone, and analogs and derivatives thereof are described, Therapeutic compositions containing the molecules, and therapeutic methods, methods of for regulating gene expression, methods for identifying modulators of the autoinducer molecules, and methods of modulating quorum sensing signalling in bacteria using the compounds of the invention are also described.
  • ItemOpen Access
    Low adenosine anti-sense oligonucleotide agent, composition, kit and treatments
    (2000-02-24) Nyce, Jonathan W.
    A composition comprises a nucleic acid comprising an oligo anti-sense to a target such as polypeptide(s) associated with an ailment afflicting lung airways, genes and mRNAs encoding them, genomic and mRNA flanking regions, intron and exon borders and all regulatory and functionally related segments of the genes and mRNAs encoding the polypeptides, their salts and mixtures. Various formulations contain a requisite carrier, and optionally other additives and biologically active agents. The agent of the invention may be prepared by selecting a target gene(s), genomic flanking region(s), RNA(s) and/or polypeptide(s) associated with a disease(s) or condition(s) afflicting lung airways, obtaining the sequence of the mRNA(s) corresponding to the target gene(s) and/or genomic flanking region(s), and/or RNAs encoding the target polypeptide(s), selecting at least one segment of the mRNA which may be up to 60 % free of thymidine (T) and synthesizing one or more anti-sense oligonucleotide(s) to the mRNA segments which are free of adenosine (A) by substituting a universal base for A when present in the oligonucleotide. The agent may be prepared by selection of target nucleic acid sequences with GC running stretches, which have low T content, and by optionally replacing A in the anti-sense oligonucleotides with a AUniversal base@. The agent, composition and formulations are used for prophylactic, preventive and therapeutic treatment of ailments associated with impaired respiration, allergy(ies) and/or inflammation, such as pulmonary vasoconstriction, inflammation, allergies, asthma, impeded respiration, lung pain, cystic fibrosis, bronchoconstriction, pulmonary hypertension and bronchoconstriction, chronic bronchitis, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), ischemic conditions including ischemia itself, and cancers such as leukemias, lymphomas, carcinomas, and the like, e.g. colon cancer, breast cancer, pancreatic cancer, lung cancer, hepatocellular carcinoma, kidney cancer, melanoma, hepatic metastasis, etc., as well as all types of cancers with may metastasize or have metastasized to the lung(s), including breast and prostate cancer. The present treatment is suitable for administration in combination with other treatments, e.g. before, during and after other treatments, including radiation, chemotherapy, antibody therapy and surgery, among others. The present agent is effectively administered preventatively, prophylactically or therapeutically by itself for conditions without known therapies, or as a substitute for, or in conjunction with, other therapies exhibiting undesirable side effects. The treatment of this invention may be administered directly into the respiratory system of a subject, so that the agent has direct access to the airways and the lungs.
  • ItemOpen Access
    Relationship of N-glycans to Neuronal Dysfunction
    (East Carolina University, 2019-06-07) Whitman, Austin A
    N-glycosylation is important in regulating protein activity and serves several vital roles contributing to protein folding, protein assembly, stability, and interactions. Changes in branching of N-glycans are linked to development and maintenance of multicellular organisms. All N-glycans share a common core sugar sequence and are classified as three major types: oligomannose, complex, and hybrid. Here the aim is to determine the roles N-glycans have in cell behavior, as well as, vertebrae development. To characterize the contribution of complex N-glycans to neuroblastoma (NB), CRISPR-Cas9 technology was employed to silence the Mgat2 gene in a NB cell line. Mgat2 encodes for GlcNAcT-II, N-acetylglucosaminyltransferase-II, responsible for converting hybrid type N-glycans to complex type. Lectin binding studies were conducted to support the successful knockout of Mgat2. Electrophoretic mobility shifts of Kv3.1, a voltage-gated K+ channel, supports that N220 and N229, possess complex N-glycans in our wild type cell line and hybrid N-glycans in our N-glycosylation mutant cell line. Wound healing, anchorage-independent growth, and MMP-2 expression studies found that the loss of Mgat2 resulted in decreased tumorigenicity. Mgat1b was silenced in zebrafish to study how increased oligomannose N-glycans affect vertebrae development. Magt1b encodes for GlcNAct-I, responsible for converting oligomannose N-glycans to hybrid type, which in turn form complex N-glycans. DNA sequencing and various genotyping of zebrafish generations support the knockout of Mgat1b. The loss of Mgat1b resulted in decreased embryo viability but also increased the number of eggs spawned. Overall, this data reveals that complex N-glycans contribute to an increase in NB tumorigenicity, and also, increased levels of oligomannose N-glycans impede development of embryos in vertebrae.
  • ItemOpen Access
    Docosahexaenoic acid-derived metabolites target the B-cell driven immune response in obese mice
    (East Carolina University, 2019-06-07) Crouch, Miranda Jill
    Obesity is associated with an impaired humoral immune response. As B cells mediate the humoral immune response, they are major cellular targets in diet induced obesity models. Several studies have established that B cell function is impaired in obesity potentially due to decreased AID levels and impaired leptin signaling. Studies have also found that B cells from obese individuals secrete pro-inflammatory cytokines and that B cells seem to have a pathological role in the visceral adipose tissue in obesity. We have also established that obese mice have decreased numbers of total B cells and B cell subsets in the bone marrow compared to their control counterparts. Despite these findings, mechanisms elucidating how the B cell response is impaired are not well established. We and others have shown that specialized pro-resolving lipid mediator (SPM) precursors and SPMs are decreased in various tissues in obese humans and mice. As SPMs promote tissue homeostasis, prevent chronic inflammation, and can regulate B cell antibody production, we investigated whether deficiencies in the levels of SPM precursors and SPMs could be contributing to the impairments in B cell subsets and B cell function in obesity. We administered a cocktail of 14-HDHA/17-HDHA/PDX to obese mice for four consecutive days as these DHA-derived metabolites were decreased in the spleens of obese mice. Overall, we found that these metabolites regulate B cell numbers in various tissues in obese mice. In particular, administration of 14-HDHA/17-HDHA/PDX to obese mice rescued decrements in total B cell and B cell subset numbers in the bone marrow. Furthermore, we found that these metabolites also decreased the number of B cells in the visceral adipose tissue, which were elevated in obese mice. We hypothesize that these DHA-derived metabolites are limiting B cell recruitment to the adipose by regulating chemokine receptor interactions with their corresponding ligands as obese mice receiving the DHA-derived metabolites had decreased transcript expression of various chemokine receptors that are upregulated in obesity. Obese mice receiving the DHA-derived metabolites also had decreased levels of pathogenic IgG2c in circulation as well as decreased IgM and IgG levels in the VAT. In addition, we established that supplementation of the parent fatty acid, DHA in the diet can increase antibody production in obese mice infected with influenza. When 14-HDHA, 17-HDHA, and PDX were administered individually to lean mice infected with influenza, 14-HDHA enhanced antibody titers and the number of antibody secreting cells in the bone marrow. Interestingly, we found that SPM precursors and SPM levels were not decreased in obese female mice. The obese female mice also did not have major decrements in B cell numbers in the bone marrow and had a decreased inflammatory profile compared to their male counterparts. Overall, findings from this dissertation propose that administration of DHA-derived metabolites that are deficient in obese mice can regulate B cell numbers across various tissues, potentially modulate the pro-inflammatory B cell phenotype in the adipose tissue, and enhance antibody production in the context of influenza.