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Full-Text Articles in Medicine and Health Sciences
A Developmental Cycle Masks Output From The Circadian Oscillator Under Conditions Of Choline Deficiency In Neurospora, Mi Shi, Luis F. Larrondo, Jennifer J. Loros, Jay C. Dunlap
A Developmental Cycle Masks Output From The Circadian Oscillator Under Conditions Of Choline Deficiency In Neurospora, Mi Shi, Luis F. Larrondo, Jennifer J. Loros, Jay C. Dunlap
Dartmouth Scholarship
In Neurospora, metabolic oscillators coexist with the circadian transcriptional/translational feedback loop governed by the FRQ (Frequency) and WC (White Collar) proteins. One of these, a choline deficiency oscillator (CDO) observed in chol-1 mutants grown under choline starvation, drives an uncompensated long-period developmental cycle ( approximately 60-120 h). To assess possible contributions of this metabolic oscillator to the circadian system, molecular and physiological rhythms were followed in liquid culture under choline starvation, but these only confirmed that an oscillator with a normal circadian period length can run under choline starvation. This finding suggested that long-period developmental cycles elicited by nutritional stress …
A Novel Inhibitory Mechanism Of Mitochondrion-Dependent Apoptosis By A Herpesviral Protein, Pinghui Feng, Chengyu Liang, Young C. Shin, Xiaofei E, Weijun Zhang, Robyn Gravel, Ting-Ting Wu, Ren Sun, Edward Usherwood, Jae U. Jung
A Novel Inhibitory Mechanism Of Mitochondrion-Dependent Apoptosis By A Herpesviral Protein, Pinghui Feng, Chengyu Liang, Young C. Shin, Xiaofei E, Weijun Zhang, Robyn Gravel, Ting-Ting Wu, Ren Sun, Edward Usherwood, Jae U. Jung
Dartmouth Scholarship
Upon viral infection, cells undergo apoptosis as a defense against viral replication. Viruses, in turn, have evolved elaborate mechanisms to subvert apoptotic processes. Here, we report that a novel viral mitochondrial anti-apoptotic protein (vMAP) of murine gamma-herpesvirus 68 (gammaHV-68) interacts with Bcl-2 and voltage-dependent anion channel 1 (VDAC1) in a genetically separable manner. The N-terminal region of vMAP interacted with Bcl-2, and this interaction markedly increased not only Bcl-2 recruitment to mitochondria but also its avidity for BH3-only pro-apoptotic proteins, thereby suppressing Bax mitochondrial translocation and activation. In addition, the central and C-terminal hydrophobic regions of vMAP interacted with VDAC1. …
Characterization Of Mazfsa, An Endoribonuclease From Staphylococcus Aureus, Zhibiao Fu, Niles P. Donegan, Guido Memmi, Ambrose L. Cheung
Characterization Of Mazfsa, An Endoribonuclease From Staphylococcus Aureus, Zhibiao Fu, Niles P. Donegan, Guido Memmi, Ambrose L. Cheung
Dartmouth Scholarship
The mazEF homologs of Staphylococcus aureus, designated mazEF(sa), have been shown to cotranscribe with the sigB operon under stress conditions. In this study, we showed that MazEF(Sa), as with their Escherichia coli counterparts, compose a toxin-antitoxin module wherein MazF(Sa) leads to rapid cell growth arrest and loss in viable CFU upon overexpression. MazF(Sa) is a novel sequence-specific endoribonuclease which cleaves mRNA to inhibit protein synthesis. Using ctpA mRNA as the model substrate both in vitro and in vivo, we demonstrated that MazF(Sa) cleaves single-strand RNA preferentially at the 5' side of the first U or 3' side of the second …
Assays Of Vacuole Fusion Resolve The Stages Of Docking, Lipid Mixing, And Content Mixing, Youngsoo Jun, William Wickner
Assays Of Vacuole Fusion Resolve The Stages Of Docking, Lipid Mixing, And Content Mixing, Youngsoo Jun, William Wickner
Dartmouth Scholarship
Membrane fusion entails organelle docking and subsequent mixing of membrane bilayers and luminal compartments. We now present an in vitro assay of fusion, using yeast vacuoles bearing domains of either Fos or Jun fused to complementary halves of beta-lactamase. Upon fusion, these proteins associate to yield beta-lactamase activity. This assay complements the standard fusion assay (activation of pro-Pho8p in protease-deficient vacuoles by proteases from pho8Delta vacuoles). Both the beta-lactamase and pro-Pho8p activation assays of fusion show the same long kinetic delay between SNARE pairing and luminal compartment mixing. Lipid-mixing occurs rapidly after SNARE pairing but well before aqueous compartment mixing. …
All-Trans Retinoic Acid Mediates Enhanced T Reg Cell Growth, Differentiation, And Gut Homing In The Face Of High Levels Of Co-Stimulation, Micah J. Benson, Karina Pino-Lagos, Mario Rosemblatt, Randolph J. Noelle
All-Trans Retinoic Acid Mediates Enhanced T Reg Cell Growth, Differentiation, And Gut Homing In The Face Of High Levels Of Co-Stimulation, Micah J. Benson, Karina Pino-Lagos, Mario Rosemblatt, Randolph J. Noelle
Dartmouth Scholarship
We demonstrate that all-trans retinoic acid (RA) induces FoxP3(+) adaptive T regulatory cells (A-Tregs) to acquire a gut-homing phenotype (alpha 4 beta 7(+) CC chemokine receptor 9(+)) and the capacity to home to the lamina propria of the small intestine. Under conditions that favor the differentiation of A-Tregs (transforming growth factor-beta1 and interleukin 2) in vitro, the inclusion of RA induces nearly all activated CD4(+) T cells to express FoxP3 and greatly increases the accumulation of these cells. In the absence of RA, A-Treg differentiation is abruptly impaired by proficient antigen presenting cells or through direct co-stimulation. In the presence …
Phenotypic Alterations In Type Ii Alveolar Epithelial Cells In Cd4+ T Cell Mediated Lung Inflammation, Marcus Gereke, Lothar Gröbe, Silvia Prettin, Michael Kasper, Stefanie Deppenmeier, Achim D. Gruber, Richard I. Enelow, Jan Buer, Dunja Bruder
Phenotypic Alterations In Type Ii Alveolar Epithelial Cells In Cd4+ T Cell Mediated Lung Inflammation, Marcus Gereke, Lothar Gröbe, Silvia Prettin, Michael Kasper, Stefanie Deppenmeier, Achim D. Gruber, Richard I. Enelow, Jan Buer, Dunja Bruder
Dartmouth Scholarship
Although the contribution of alveolar type II epithelial cell (AEC II) activities in various aspects of respiratory immune regulation has become increasingly appreciated, our understanding of the contribution of AEC II transcriptosome in immunopathologic lung injury remains poorly understood. We have previously established a mouse model for chronic T cell-mediated pulmonary inflammation in which influenza hemagglutinin (HA) is expressed as a transgene in AEC II, in mice expressing a transgenic T cell receptor specific for a class II-restricted epitope of HA. Pulmonary inflammation in these mice occurs as a result of CD4+ T cell recognition of alveolar antigen. This model …
Bifa, A Cyclic-Di-Gmp Phosphodiesterase, Inversely Regulates Biofilm Formation And Swarming Motility By Pseudomonas Aeruginosa Pa14, Sherry L. Kuchma, Kimberly M. Brothers, Judith H. Merritt, Nicole T. Liberati, Frederick M. Ausubel, George A. O'Toole
Bifa, A Cyclic-Di-Gmp Phosphodiesterase, Inversely Regulates Biofilm Formation And Swarming Motility By Pseudomonas Aeruginosa Pa14, Sherry L. Kuchma, Kimberly M. Brothers, Judith H. Merritt, Nicole T. Liberati, Frederick M. Ausubel, George A. O'Toole
Dartmouth Scholarship
The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner …
Formation Of Native Prions From Minimal Components In Vitro, Nathan R. Deleault, Brent T. Harris, Judy R. Rees, Surachai Supattapone
Formation Of Native Prions From Minimal Components In Vitro, Nathan R. Deleault, Brent T. Harris, Judy R. Rees, Surachai Supattapone
Dartmouth Scholarship
The conformational change of a host protein, PrPC, into a disease-associated isoform, PrPSc, appears to play a critical role in the pathogenesis of prion diseases such as Creutzfeldt–Jakob disease and scrapie. However, the fundamental mechanism by which infectious prions are produced in neurons remains unknown. To investigate the mechanism of prion formation biochemically, we conducted a series of experiments using the protein misfolding cyclic amplification (PMCA) technique with a preparation containing only native PrPC and copurified lipid molecules. These experiments showed that successful PMCA propagation of PrPScmolecules in a purified system requires accessory polyanion molecules. In addition, we found that …
The Flagellum Of Pseudomonas Aeruginosa Is Required For Resistance To Clearance By Surfactant Protein A, Shiping Zhang, Francis X. Mccormack, Roger C. Levesque, George A. O'Toole, Gee W. Lau
The Flagellum Of Pseudomonas Aeruginosa Is Required For Resistance To Clearance By Surfactant Protein A, Shiping Zhang, Francis X. Mccormack, Roger C. Levesque, George A. O'Toole, Gee W. Lau
Dartmouth Scholarship
Surfactant protein A (SP-A) is an important lung innate immune protein that kills microbial pathogens by opsonization and membrane permeabilization. We investigated the basis of SP-A-mediated pulmonary clearance of Pseudomonas aeruginosa using genetically-engineered SP-A mice and a library of signature-tagged P. aeruginosa mutants. A mutant with an insertion into flgE, the gene that encodes flagellar hook protein, was preferentially cleared by the SP-A(+/+) mice, but survived in the SP-A(-/-) mice. Opsonization by SP-A did not play a role in flgE clearance. However, exposure to SP-A directly permeabilized and killed the flgE mutant, but not the wild-type parental strain. P. aeruginosa …
Crystal Structure Of The Vibrio Cholerae Quorum-Sensing Regulatory Protein Hapr, Rukman S. De Silva, Gabriela Kovacikova, Wei Lin, Ronald K. Taylor, Karen Skorupski, F. Jon Kull
Crystal Structure Of The Vibrio Cholerae Quorum-Sensing Regulatory Protein Hapr, Rukman S. De Silva, Gabriela Kovacikova, Wei Lin, Ronald K. Taylor, Karen Skorupski, F. Jon Kull
Dartmouth Scholarship
Quorum sensing in Vibrio cholerae involves signaling between two-component sensor protein kinases and the response regulator LuxO to control the expression of the master regulator HapR. HapR, in turn, plays a central role in regulating a number of important processes, such as virulence gene expression and biofilm formation. We have determined the crystal structure of HapR to 2.2-Å resolution. Its structure reveals a dimeric, two-domain molecule with an all-helical structure that is strongly conserved with members of the TetR family of transcriptional regulators. The N-terminal DNA-binding domain contains a helix-turn-helix DNA-binding motif and alteration of certain residues in this domain …
P53 Activation By Knockdown Technologies, Mara E. Robu, Jon D. Larson, Aidas Nasevicius, Soraya Beiraghi, Charles Brenner
P53 Activation By Knockdown Technologies, Mara E. Robu, Jon D. Larson, Aidas Nasevicius, Soraya Beiraghi, Charles Brenner
Dartmouth Scholarship
Morpholino phosphorodiamidate antisense oligonucleotides (MOs) and short interfering RNAs (siRNAs) are commonly used platforms to study gene function by sequence-specific knockdown. Both technologies, however, can elicit undesirable off-target effects. We have used several model genes to study these effects in detail in the zebrafish, Danio rerio. Using the zebrafish embryo as a template, correct and mistargeting effects are readily discernible through direct comparison of MO-injected animals with well-studied mutants. We show here indistinguishable off-targeting effects for both maternal and zygotic mRNAs and for both translational and splice-site targeting MOs. The major off-targeting effect is mediated through p53 activation, as detected …
Trans-Snare Complex Assembly And Yeast Vacuole Membrane Fusion, Kevin M. Collins, William T. Wickner
Trans-Snare Complex Assembly And Yeast Vacuole Membrane Fusion, Kevin M. Collins, William T. Wickner
Dartmouth Scholarship
cis-SNARE complexes (anchored in one membrane) are disassembled by Sec17p (α-SNAP) and Sec18p (NSF), permitting the unpaired SNAREs to assemble in trans. We now report a direct assay of trans-SNARE complex formation during yeast vacuole docking. SNARE complex assembly and fusion is promoted by high concentrations of the SNARE Vam7p or Nyv1p or by addition of HOPS (homotypic fusion and vacuole protein sorting), a Ypt7p (Rab)-effector complex with a Sec1/Munc18-family subunit. Inhibitors that target Ypt7p, HOPS, or key regulatory lipids prevent trans-SNARE complex assembly and ensuing fusion. Strikingly, the lipid ligand MED (myristoylated alanine-rich C kinase substrate effector domain) or …
The Pseudomonas Aeruginosa Secreted Protein Pa2934 Decreases Apical Membrane Expression Of The Cystic Fibrosis Transmembrane Conductance Regulator, Daniel P. Maceachran, Siying Ye, Jennifer M. Bomberger, Deborah A. Hogan, Agnieszka Swiatecka-Urban, Bruce Stanton, George A. O'Toole
The Pseudomonas Aeruginosa Secreted Protein Pa2934 Decreases Apical Membrane Expression Of The Cystic Fibrosis Transmembrane Conductance Regulator, Daniel P. Maceachran, Siying Ye, Jennifer M. Bomberger, Deborah A. Hogan, Agnieszka Swiatecka-Urban, Bruce Stanton, George A. O'Toole
Dartmouth Scholarship
We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2934 [corrected], and we have named the gene cif, for CFTR inhibitory factor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression …
Cpg Hypomethylation In A Large Domain Encompassing The Embryonic Β-Like Globin Genes In Primitive Erythrocytes, Mei Hsu, Rodwell R. Mabaera, Christopher H. Lowrey, David I. K. Martin, Steven Fiering
Cpg Hypomethylation In A Large Domain Encompassing The Embryonic Β-Like Globin Genes In Primitive Erythrocytes, Mei Hsu, Rodwell R. Mabaera, Christopher H. Lowrey, David I. K. Martin, Steven Fiering
Dartmouth Scholarship
There is little evidence addressing the role of CpG methylation in transcriptional control of genes that do not contain CpG islands. This is reflected in the ongoing debate about whether CpG methylation merely suppresses retroelements or if it also plays a role in developmental and tissue-specific gene regulation. The genes of the β-globin locus are an important model of mammalian developmental gene regulation and do not contain CpG islands. We have analyzed the methylation status of regions in the murine β-like globin locus in uncultured primitive and definitive erythroblasts and other cultured primary and transformed cell types. A large (∼20-kb) …
Membrane Association And Multimerization Of Tcpt, The Cognate Atpase Ortholog Of The Vibrio Cholerae Toxin-Coregulated-Pilus Biogenesis Apparatus, Shital A. Tripathi, Ronald K. Taylor
Membrane Association And Multimerization Of Tcpt, The Cognate Atpase Ortholog Of The Vibrio Cholerae Toxin-Coregulated-Pilus Biogenesis Apparatus, Shital A. Tripathi, Ronald K. Taylor
Dartmouth Scholarship
The toxin-coregulated pilus (TCP) is one of the major virulence factors of Vibrio cholerae. Biogenesis of this type 4 pilus (Tfp) requires a number of structural components encoded by the tcp operon. TcpT, the cognate putative ATPase, is required for TCP biogenesis and all TCP-mediated functions. We studied the stability and localization of TcpT in cells containing in-frame deletions in each of the tcp genes. TcpT was detectable in each of the biogenesis mutants except the ΔtcpT strain. TcpT was localized to the inner membrane (IM) in a TcpR-dependent manner. TcpR is a predicted bitopic inner membrane protein …
The Nestin Progenitor Lineage Is The Compartment Of Origin For Pancreatic Intraepithelial Neoplasia, Catherine Carriere, Elliot S. Seeley, Tobias Goetze, Daniel S. Longnecker, Murray Korc
The Nestin Progenitor Lineage Is The Compartment Of Origin For Pancreatic Intraepithelial Neoplasia, Catherine Carriere, Elliot S. Seeley, Tobias Goetze, Daniel S. Longnecker, Murray Korc
Dartmouth Scholarship
To determine the cell compartment in which initial oncogenic mutations occur in pancreatic ductal adenocarcinoma (PDAC), we generated a mouse model in which endogenous expression of mutated Kras (Kras(G12D)) was initially directed to a population of pancreatic exocrine progenitors characterized by the expression of Nestin. Targeting of oncogenic Kras to such a restricted cell compartment was sufficient for the formation of pancreatic intraepithelial neoplasias (PanINs), putative precursors to PDAC. PanINs appeared with the same grade and frequency as observed when Kras(G12D) was targeted to the whole pancreas by a Pdx1-driven Cre recombinase strategy. Thus, the Nestin cell lineage is highly …
Transgenic Cyclin E Triggers Dysplasia And Multiple Pulmonary Adenocarcinomas, Yan Ma, Steven Fiering, Candice Black, Xi Liu, Ziqiang Yuan, Vincent A. Memoli, David J. Robbins, Heather A. Bentley, Gregory J. Tsongalis, Eugene Demidenko, Sarah J. Freemantle, Ethan Dmitrovsky
Transgenic Cyclin E Triggers Dysplasia And Multiple Pulmonary Adenocarcinomas, Yan Ma, Steven Fiering, Candice Black, Xi Liu, Ziqiang Yuan, Vincent A. Memoli, David J. Robbins, Heather A. Bentley, Gregory J. Tsongalis, Eugene Demidenko, Sarah J. Freemantle, Ethan Dmitrovsky
Dartmouth Scholarship
Cyclin E is a critical G(1)-S cell cycle regulator aberrantly expressed in bronchial premalignancy and lung cancer. Cyclin E expression negatively affects lung cancer prognosis. Its role in lung carcinogenesis was explored. Retroviral cyclin E transduction promoted pulmonary epithelial cell growth, and small interfering RNA targeting of cyclin E repressed this growth. Murine transgenic lines were engineered to mimic aberrant cyclin E expression in the lung. Wild-type and proteasome degradation-resistant human cyclin E transgenic lines were independently driven by the human surfactant C (SP-C) promoter. Chromosome instability (CIN), pulmonary dysplasia, sonic hedgehog (Shh) pathway activation, adenocarcinomas, and metastases occurred. Notably, …
Inverse Regulation Of Biofilm Formation And Swarming Motility By Pseudomonas Aeruginosa Pa14, Nicky C. Caiazza, Judith H. Merritt, Kimberly M. Brothers, George A. O'Toole
Inverse Regulation Of Biofilm Formation And Swarming Motility By Pseudomonas Aeruginosa Pa14, Nicky C. Caiazza, Judith H. Merritt, Kimberly M. Brothers, George A. O'Toole
Dartmouth Scholarship
We previously reported that SadB, a protein of unknown function, is required for an early step in biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa. Here we report that a mutation in sadB also results in increased swarming compared to the wild-type strain. Our data are consistent with a model in which SadB inversely regulates biofilm formation and swarming motility via its ability both to modulate flagellar reversals in a viscosity-dependent fashion and to influence the production of the Pel exopolysaccharide. We also show that SadB is required to properly modulate flagellar reversal rates via chemotaxis cluster IV (CheIV cluster). …
The Myc Transactivation Domain Promotes Global Phosphorylation Of The Rna Polymerase Ii Carboxy-Terminal Domain Independently Of Direct Dna Binding, Victoria H. Cowling, Michael D. Cole
The Myc Transactivation Domain Promotes Global Phosphorylation Of The Rna Polymerase Ii Carboxy-Terminal Domain Independently Of Direct Dna Binding, Victoria H. Cowling, Michael D. Cole
Dartmouth Scholarship
Myc is a transcription factor which is dependent on its DNA binding domain for transcriptional regulation of target genes. Here, we report the surprising finding that Myc mutants devoid of direct DNA binding activity and Myc target gene regulation can rescue a substantial fraction of the growth defect in myc−/− fibroblasts. Expression of the Myc transactivation domain alone induces a transcription-independent elevation of the RNA polymerase II (Pol II) C-terminal domain (CTD) kinases cyclin-dependent kinase 7 (CDK7) and CDK9 and a global increase in CTD phosphorylation. The Myc transactivation domain binds to the transcription initiation sites of these promoters …