Medicine and Health Sciences Commons^™

Cyclin-Dependent Kinase Inhibitor P1446a Induces Apoptosis In A Jnk/P38 Mapk-Dependent Manner In Chronic Lymphocytic Leukemia B-Cells, Cody Paiva, J. Claire Godbersen, Ryan S. Soderquist, Taylor Rowland, Sumner Kilmarx

Dartmouth Scholarship

CDK (cyclin-dependent kinase) inhibitors have shown remarkable activity in CLL, where its efficacy has been linked to inhibition of the transcriptional CDKs (7 and 9) and deregulation of RNA polymerase and short-lived pro-survival proteins such as MCL1. Furthermore, ER (endoplasmic reticulum) stress has been implicated in CDK inhibition in CLL. Here we conducted a pre-clinical study of a novel orally active kinase inhibitor P1446A in CLL B-cells. P1446A inhibited CDKs at nanomolar concentrations and induced rapid apoptosis of CLL cells in vitro, irrespective of chromosomal abnormalities or IGHV mutational status. Apoptosis preceded inactivation of RNA polymerase, and was accompanied by …

Site-Specific Mutation Of The Sensor Kinase Gras In Staphylococcus Aureus Alters The Adaptive Response To Distinct Cationic Antimicrobial Peptides, Ambrose L. Cheung, Arnold S. Bayer, Michael R. Yeaman, Yan Q. Xiong, Alan J. Waring, Guido Memmi, Niles Donegan

Dartmouth Scholarship

The Staphylococcus aureus two-component regulatory system, GraRS, is involved in resistance to killing by distinct host defense cationic antimicrobial peptides (HD-CAPs). It is believed to regulate downstream target genes such as mprF and dltABCD to modify the S. aureus surface charge. However, the detailed mechanism(s) by which the histidine kinase, GraS, senses specific HD-CAPs is not well defined. Here, we studied a well-characterized clinical methicillin-resistant S. aureus (MRSA) strain (MW2), its isogenic graS deletion mutant (ΔgraS strain), a nonameric extracellular loop mutant (ΔEL strain), and four residue-specific ΔEL mutants (D37A, P39A, P39S, and D35G D37G D41G strains). The ΔgraS and …

Influence Networks Based On Coexpression Improve Drug Target Discovery For The Development Of Novel Cancer Therapeutics, Nadia M. Penrod, Jason H. Moore

Dartmouth Scholarship

Background: Thedemandfornovelmolecularlytargeteddrugswillcontinuetoriseaswemoveforwardtowardthe goal of personalizing cancer treatment to the molecular signature of individual tumors. However, the identification of targets and combinations of targets that can be safely and effectively modulated is one of the greatest challenges facing the drug discovery process. A promising approach is to use biological networks to prioritize targets based on their relative positions to one another, a property that affects their ability to maintain network integrity and propagate information-flow. Here, we introduce influence networks and demonstrate how they can be used to generate influence scores as a network-based metric to rank genes as potential drug targets. …

Importance Of Bacillithiol In The Oxidative Stress Response Of Staphylococcus Aureus, Ana C. Posada, Stacey L. Kolar, Renata G. Dusi, Patrica Francois

Dartmouth Scholarship

In Staphylococcus aureus, the low-molecular-weight thiol called bacillithiol (BSH), together with cognate S-transferases, is believed to be the counterpart to the glutathione system of other organisms. To explore the physiological role of BSH in S. aureus, we constructed mutants with the deletion of bshA (sa1291), which encodes the glycosyltransferase that catalyzes the first step of BSH biosynthesis, and fosB (sa2124), which encodes a BSH-S-transferase that confers fosfomycin resistance, in several S. aureus strains, including clinical isolates. Mutation of fosB or bshA caused a 16- to 60-fold reduction in fosfomycin resistance in these S. aureus strains. High-pressure liquid chromatography analysis, which …

Selective Impact Of Hiv Disease Progression On The Innate Immune System In The Human Female Reproductive Tract, Timothy Lahey, Mimi Ghosh, John V. Fahey, Zheng Shen, Lucy R. Mukura, Yan Song, Susan Cu-Uvin, Kenneth H. Mayer, Peter F. Wright, John C. Kappes, Christina Ochsenbauer, Charles R. Wira

Dartmouth Scholarship

Background: We have previously demonstrated intrinsic anti-HIV activity in cervicovaginal lavage (CVL) from HIV-infected women with high CD4 counts and not on antiretroviral therapy. However, the impact of HIV disease progression on CVL innate immune responses has not been delineated.

Methods: CVL from 57 HIV-infected women not on antiretroviral therapy were collected by washing the cervicovaginal area with 10 ml of sterile normal saline. We characterized subject HIV disease progression by CD4 count strata: >500 cells/µl, 200-500 cells/µl, or <200 cells/µl of blood. To assess CVL anti-HIV activity, we incubated TZM-bl cells with HIV plus or minus CVL. Antimicrobials, cytokines, chemokines and anti-gp160 HIV IgG antibodies were measured by ELISA and Luminex.

Β-Lactams Interfering With Pbp1 Induce Panton-Valentine Leukocidin Expression By Triggering Sara And Rot Global Regulators Of Staphylococcus Aureus, Oana Dumitrescu, Priya Choudhury, Sandrine Boisset, Cedric Badiou, Michele Bes, Yvonne Benito, Christiane Wolz, Francois Vandenesch, Jerome Etienne, Ambrose L. Cheung

Dartmouth Scholarship

Previous articles reported that beta-lactam antibiotics increase the expression of Staphylococcus aureus Panton-Valentine leukocidin (PVL) by activating its transcription. We investigated the mechanisms underlying the inductor effect of beta-lactams on PVL expression by determining targets and regulatory pathways possibly implicated in this process. We measured PVL production in the presence of oxacillin (nonselective), imipenem (penicillin-binding protein 1 [PBP1] selective), cefotaxime (PBP2 s

Cif Is Negatively Regulated By The Tetr Family Repressor Cifr, Daniel P. Maceachran, Bruce A. Stanton, George A. O'Toole

Dartmouth Scholarship

We previously reported that the novel Pseudomonas aeruginosa toxin Cif is capable of decreasing apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR). We further demonstrated that Cif is capable of degrading the synthetic epoxide hydrolase (EH) substrate S-NEPC [(2S,3S)-trans-3-phenyl-2-oxiranylmethyl 4-nitrophenol carbonate], suggesting that Cif may be reducing apical membrane expression of CFTR via its EH activity. Here we report that Cif is capable of degrading the xenobiotic epoxide epibromohydrin (EBH) to its vicinal diol 3-bromo-1,2-propanediol. We also demonstrate that this epoxide is a potent inducer of cif gene expression. We show that the predicted TetR family transcriptional …

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 …

Innate Antiviral Response Targets Hiv-1 Release By The Induction Of Ubiquitin-Like Protein Isg15, Atsushi Okumura, Gengshi Lu, Ian Pitha-Rowe, Paula M. Pitha

Dartmouth Scholarship

The goal of this study was to elucidate the molecular mechanism by which type I IFN inhibits assembly and release of HIV-1 virions. Our study revealed that the IFN-induced ubiquitin-like protein ISG15 mimics the IFN effect and inhibits release of HIV-1 virions without having any effect on the synthesis of HIV-1 proteins in the cells. ISG15 expression specifically inhibited ubiquitination of Gag and Tsg101 and disrupted the interaction of the Gag L domain with Tsg101, but conjugation of ISG15 to Gag or Tsg101 was not detected. The inhibition of Gag-Tsg101 interaction was also detected in HIV-1 infected, IFN-treated cells. Elimination …

Tcpf Is A Soluble Colonization Factor And Protective Antigen Secreted By El Tor And Classical O1 And O139 Vibrio Cholerae Serogroups, Thomas J. Kirn, Ronald K. Taylor

Dartmouth Scholarship

Vibrio cholerae causes diarrhea by colonizing the human small bowel and intoxicating epithelial cells. Colonization is a required step in pathogenesis, and strains defective for colonization are significantly attenuated. The best-characterized V. cholerae colonization factor is the toxin-coregulated pilus (TCP). It has been demonstrated that TCP is required for V. cholerae colonization in both humans and mice. TCP enhances bacterial interactions that allow microcolony formation and thereby promotes survival in the intestine. We have recently discovered that the TCP biogenesis apparatus also serves as a secretion system, mediating the terminal step in the extracellular secretion pathway of TcpF. TcpF was …

Effects Of Estradiol On Lipopolysaccharide And Pam3cys Stimulation Of Ccl20/Macrophage Inflammatory Protein 3 Alpha And Tumor Necrosis Factor Alpha Production By Uterine Epithelial Cells In Culture, Mardi A. Crane-Godreau, Charles R. Wira

Dartmouth Scholarship

We have previously demonstrated that rat uterine epithelial cells (UEC) produce CCL20/macrophage inflammatory protein 3 alpha (MIP3alpha) and tumor necrosis factor alpha (TNF-alpha) in response to live and heat-killed Escherichia coli and to the pathogen-associated molecular patterns (PAMP) lipopolysaccharide (LPS) and Pam3Cys. To determine whether estradiol (E2) modulates PAMP-induced CCL20/MIP3alpha and TNF-alpha secretion, primary cultures of rat UEC were incubated with E2 for 24 h and then treated with LPS or Pam3Cys or not treated for an additional 12 h. E2 inhibited the constitutive secretion of TNF-alpha and CCL20/MIP3alpha into culture media. Interestingly, E2 pretreatment enhanced CCL20/MIP3alpha secretion due to …

Probucol Prevents Early Coronary Heart Disease And Death In The High-Density Lipoprotein Receptor Sr-Bi/Apolipoprotein E Double Knockout Mouse, Anne Braun, Songwen Zhang, Helena E. Miettinen, Shamsah Ebrahim, Teresa M. Holm, Eliza Vasile, Mark J. Post

Dartmouth Scholarship

Mice with homozygous null mutations in the high-density lipoprotein receptor SR-BI (scavenger receptor class B, type I) and apolipoprotein E genes fed a low-fat diet exhibit a constellation of pathologies shared with human atherosclerotic coronary heart disease (CHD): hypercholesterolemia, occlusive coronary atherosclerosis, myocardial infarctions, cardiac dysfunction (heart enlargement, reduced systolic function and ejection fraction, and ECG abnormalities), and premature death (mean age 6 weeks). They also exhibit a block in RBC maturation and abnormally high plasma unesterified-to-total cholesterol ratio (0.8) with associated abnormal lipoprotein morphology (lamellar/vesicular and stacked discoidal particles reminiscent of those in lecithin/cholesterol acyltransferase deficiency and cholestasis). Treatment …

Regulation And Localization Of Endogenous Human Tristetraprolin, Anna-Marie Fairhurst, John E. Connolly, Katharine A Hintz, Nicolas J Goulding

Dartmouth Scholarship

Tumor necrosis factor (TNF) has been implicated in the development and pathogenicity of infectious diseases and autoimmune disorders, such as septic shock and arthritis. The zinc-finger protein tristetraprolin (TTP) has been identified as a major regulator of TNF biosynthesis. To define its intracellular location and examine its regulation of TNF, a quantitive intracellular staining assay specific for TTP was developed. We establish for the first time that in peripheral blood leukocytes, express

Evaluation Of A Tetracycline-Inducible Promoter In Staphylococcus Aureus In Vitro And In Vivo And Its Application In Demonstrating The Role Of Sigb In Microcolony Formation, B. T. Bateman, N. P. Donegan, T. M. Jarry, M. Palma

Dartmouth Scholarship

An inducible promoter system provides a powerful tool for studying the genetic basis for virulence. A variety of inducible systems have been used in other organisms, including pXyl-xylR-inducible promoter, the pSpac-lacI system, and the arabinose-inducible P_BAD promoter, but each of these systems has limitations in its application to Staphylococcus aureus. In this study, we demonstrated the efficacy of a tetracycline-inducible promoter system in inducing gene expression in S. aureus in vitro and inside epithelial cells as well as in an animal model of infection. Using the xyl/tetO promoter::gfp_uvr fusion carried on a shuttle …

A Thyroid Hormone-Regulated Gene In Xenopus Laevis Encodes A Type Iii Iodothyronine 5-Deiodinase., Donald L. St Germain, Robert Schwartzman, Walburga Croteau, Akira Kanamori, Zhou Wang, Donald D. Brown, Valerie Galton

Dartmouth Scholarship

The type III iodothyronine 5-deiodinase metabolizes thyroxine and 3,5,3'-triiodothyronine to inactive metabolites by catalyzing the removal of iodine from the inner ring. The enzyme is expressed in a tissue-specific pattern during particular stages of development in amphibia, birds, and mammals. Recently, a PCR-based subtractive hybridization technique has been used to isolate cDNAs prepared from Xenopus laevis tadpole tail mRNA that represent genes upregulated by thyroid hormone during metamorphosis. Sequence analysis of one of these cDNAs (XL-15) revealed regions of homology to the mRNA encoding the rat type I (outer ring) 5'-deiodinase, including a conserved UGA codon that encodes selenocysteine in …

Impairment Of The Cellular Immune Response In Acute Murine Toxoplasmosis: Regulation Of Interleukin 2 Production And Macrophage-Mediated Inhibitory Effects., Sakhina Haque, Imtiaz Khan, Azizul Haque, Lloyd Kasper

Dartmouth Scholarship

Depression of the cellular immune response to Toxoplasma gondii has been reported in both mice and humans. The present study was undertaken to determine the kinetics and mechanism of the observed downregulation of interleukin 2 (IL-2) production during experimental murine toxoplasmosis. For these investigations, the cell-mediated immune response to the wild type (PTg) was compared with that to the less-virulent mutant parasite (PTgB), which is deficient in the major surface antigen, p30 (SAG-1). Spleen cells from infected A/J mice failed to proliferate in response to Toxoplasma antigens during the first week of infection. Both PTg- and PTgB-infected A/J mice exhibited …