Francisella tularensis 2-C-Methyl-D-Erythritol 4-Phosphate Cytidylyltransferase: Kinetic Characterization and Phosphoregulation

Deliberate and natural outbreaks of infectious disease, the prevalence of antibiotic resistant strains, and the ease by which antibiotic resistant bacteria can be intentionally engineered all underscore the necessity of effective vaccines and continued development of novel antimicrobial/antiviral therapeutics. Isoprenes, a group of molecules fundamentally involved in a variety of crucial biological functions, are derived from either the mevalonic acid (MVA) or methylerythritol phosphate (MEP) pathway. While mammals utilize the MVA pathway, many bacteria utilize the MEP pathway, highlighting the latter as an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP cytidylyltransferase, a MEP pathway enzyme and potential target for antibiotic development. Size exclusion chromatography indicates the protein exists as a dimer in solution. Enzyme assays produced an apparent , , , , and a . The enzyme exhibits a strict preference for Mg+2 as a divalent cation and CTP as the nucleotide. Titanium dioxide chromatography-tandem mass spectrometry identified Thr141 as a site of phosphorylation. T141D and T141E site-directed mutants are catalytically inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP cytidylyltransferase is an excellent target for the development of novel antibiotics against F. tularensis

Solid-Phase Microextraction and the Human Fecal VOC Metabolome

The diagnostic potential and health implications of volatile organic compounds (VOCs) present in human feces has begun to receive considerable attention. Headspace solid-phase microextraction (SPME) has greatly facilitated the isolation and analysis of VOCs from human feces. Pioneering human fecal VOC metabolomic investigations have utilized a single SPME fiber type for analyte extraction and analysis. However, we hypothesized that the multifarious nature of metabolites present in human feces dictates the use of several diverse SPME fiber coatings for more comprehensive metabolomic coverage. We report here an evaluation of eight different commercially available SPME fibers, in combination with both GC-MS and GC-FID, and identify the 50/30 µm CAR-DVB-PDMS, 85 µm CAR-PDMS, 65 µm DVB-PDMS, 7 µm PDMS, and 60 µm PEG SPME fibers as a minimal set of fibers appropriate for human fecal VOC metabolomics, collectively isolating approximately 90% of the total metabolites obtained when using all eight fibers. We also evaluate the effect of extraction duration on metabolite isolation and illustrate that ex vivo enteric microbial fermentation has no effect on metabolite composition during prolonged extractions if the SPME is performed as described herein

1961

My Ideal Course, Underwater, U.S.A. (page 1) From the Editor (3) Turf Management Club News (3) Quotes from 1961 Seniors (4) The United States Most Western Owned Golf Course: Armed Forces Golf Course, Guam (5) Turf Majors Participate in Horticultural Show (7) Picture - G.C.S.A Scholarships Awarded to Three Turf Seniors (8) Picture - Stockbridge - Majors in turf Management (9) Opportunity and Education (10) The Most Outstanding Turf Senior for the Year - 1961 (11) How We Prepare Our Greens Before Topdressing (12) An Inexpensive Cure for Weeds and Poa Annua (13) Watering (14) Picture - Honorary Members of the Turf Management Club (16) Picture - Graduates of Winter School for Turf Managers - 1961 (17) Welcome Speech by Narry Sperandio (A-1) Handle with Care by Dr. Ellsworth H. Wheeler (A-2) Current Ideas on Green Construction - Panel Discussion (A-4) Automatic Systems for Watering by Robert F. Harper (A-14) History of Golf Course Architecture by Geoffrey S. Cornish (A-22) Effect of Nutrition on Turf Diseases by Dr. Houston B. Couch Turf Disease Control and Use of Fungicides by Dr. R. J. Lukens Trees and Tree Care by Gordon S. King (A-38) Arsenical Toxicity by Dr. C. R. Skogley (A-41) Soil Reaction to Arsenical Compounds by Joseph E. Steckel Brush Control For the Golf Course by Dr. William I. Boyd (A-51) Massachusetts Highway Herbicide Program by Joseph L. Beasley (A-54) General Turf (Alternate Session): Observations on Highway Turf Establishment & Maintenance by E.F. Button (A-62) Pre-emerge Chemicals for the Control of Crabgrass by Dr. John R. Havis, John M. Zak & Joseph Troll (A-70) Root Growth of Turf Grasses as Affected by Different heights of Cut and Nutrient Levels by Evangel J. Bredakis (A-71) The Use of Sod by Daniel Pellegrino (A-72

Mutations and Deregulation of Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Cascades Which Alter Therapy Response

The Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascades are often activated by genetic alterations in upstream signaling molecules such as receptor tyrosine kinases (RTK). Certain components of these pathways, RAS, NF1, BRAF, MEK1, DUSP5, PP2A, PIK3CA, PIK3R1, PIK3R4, PIK3R5, IRS4, AKT, NFKB1, MTOR, PTEN, TSC1, and TSC2 may also be activated/inactivated by mutations or epigenetic silencing. Upstream mutations in one signaling pathway or even in downstream components of the same pathway can alter the sensitivity of the cells to certain small molecule inhibitors. These pathways have profound effects on proliferative, apoptotic and differentiation pathways. Dysregulation of components of these cascades can contribute to: resistance to other pathway inhibitors, chemotherapeutic drug resistance, premature aging as well as other diseases. This review will first describe these pathways and discuss how genetic mutations and epigenetic alterations can result in resistance to various inhibitors