Lessons for the DoD from the nonprofit sector

MBA Professional ReportThe objective of this MBA project is to examine the nonprofit sector as a source of lessons learned for the federal government and the Department of Defense. This paper provides a characterization of the nonprofit and public sectors to identify the attributes common to both sectors. Utilizing the similarities between the sectors, we make the case for the nonprofit sector as an untapped source of lessons learned for the public sector and DoD. We then demonstrate our case for nonprofit organizations as a source of lessons learned using the American Red Cross as an example as it shares many of the same challenges as the federal government.http://archive.org/details/lessonsfordodfro109459895Lieutenant Commander, Supply Corps, US NavyApproved for public release; distribution is unlimited

Identification and Characterization of Inhibitors of the Aminoglycoside Resistance Acetyltransferase Eis from Mycobacterium tuberculosis

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/89566/1/73_ftp.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/89566/2/cmdc_201100332_sm_miscellaneous_information.pd

Political vetting of community work in Northern Ireland

In April 1990 the Community Workers' Co-op and the Northern Ireland Council for Voluntary Action organised a very well attended, broad based conference at Queen's University to discuss issues arising from the Government's policy of political vetting and it's effect on community work. It was quite clear from the conference that the effects of political vetting were much wider than those dozen or so groups directly penalised. This is primarily a report of the conference but also includes an update of the latest victim of this policy the Irish language group Glór na nGael

Generating Enzyme and Radical‐Mediated Bisubstrates as Tools for Investigating Gcn5‐Related N‐Acetyltransferases

Gcn5‐related N‐acetyltransferases (GNATs) are found in all kingdoms of life and catalyze important acyl transfer reactions in diverse cellular processes. While many 3D structures of GNATs have been determined, most do not contain acceptor substrates in their active sites. To expand upon existing crystallographic strategies for improving acceptor‐bound GNAT structures, we synthesized peptide substrate analogs and reacted them with CoA in PA4794 protein crystals. We found two separate mechanisms for bisubstrate formation: (a) a novel X‐ray induced radical‐mediated alkylation of CoA with an alkene peptide and (b) direct alkylation of CoA with a halogenated peptide. Our approach is widely applicable across the GNAT superfamily and can be used to improve the success rate of obtaining liganded structures of other acyltransferases

Dynamics-function relationship in the catalytic domains of N-terminal acetyltransferases

N-terminal acetyltransferases (NATs) belong to the superfamily of acetyltransferases. They are enzymes catalysing the transfer of an acetyl group from acetyl coenzyme A to the N-terminus of polypeptide chains. N-terminal acetylation is one of the most common protein modifications. To date, not much is known on the molecular basis for the exclusive substrate specificity of NATs. All NATs share a common fold called GNAT. A characteristic of NATs is the β6β7 hairpin loop covering the active site and forming with the α1α2 loop a narrow tunnel surrounding the catalytic site in which cofactor and polypeptide meet and exchange an acetyl group. We investigated the dynamics-function relationships of all available structures of NATs covering the three domains of Life. Using an elastic network model and normal mode analysis, we found a common dynamics pattern conserved through the GNAT fold; a rigid V-shaped groove formed by the β4 and β5 strands and splitting the fold in two dynamical subdomains. Loops α1α2, β3β4 and β6β7 all show clear displacements in the low frequency normal modes. We characterized the mobility of the loops and show that even limited conformational changes of the loops along the low-frequency modes are able to significantly change the size and shape of the ligand binding sites. Based on the fact that these movements are present in most low-frequency modes, and common to all NATs, we suggest that the α1α2 and β6β7 loops may regulate ligand uptake and the release of the acetylated polypeptide.publishedVersio

New qnr Gene Cassettes Associated with Superintegron Repeats in Vibrio cholerae O1

A novel qnr determinant emerged in ciprofloxacin-resistant Vibrio cholerae O1 from the Amazon region of Brazil. This qnrVC1 was in a typical class 1 integron. Its attC showed 89% identity with V. parahaemolyticus superintegron repeats. Analysis showed V. cholerae O1 carrying qnrVC2 associated with a V. cholerae superintegron repeat

Biochemical and Thermodynamic Analyses of Salmonella enterica Pat, a Multidomain, Multimeric Nε-Lysine Acetyltransferase Involved in Carbon and Energy Metabolism

In the bacterium Salmonella enterica, the CobB sirtuin protein deacetylase and the Gcn5-related Nε-acetyltransferase (GNAT) Pat control carbon utilization and metabolic flux via Nε-lysine acetylation/deacetylation of metabolic enzymes. To date, the S. enterica Pat (SePat) acetyltransferase has not been biochemically characterized. Here we report the kinetic and thermodynamic characterization of the SePat enzyme using two of its substrates, acetyl coenzyme A (Ac-CoA) synthetase (Acs; AMP forming, EC 6.2.1.1) and Ac-CoA. The data showed typical Michaelis-Menten kinetic behavior when Ac-CoA was held at a saturating concentration while Acs was varied, and a sigmoidal kinetic behavior was observed when Acs was saturating and the Ac-CoA concentration was varied. The observation of sigmoidal kinetics and positive cooperativity for Ac-CoA is an unusual feature of GNATs. Results of isothermal titration calorimetry (ITC) experiments showed that binding of Ac-CoA to wild-type SePat produced a biphasic curve having thermodynamic properties consistent with two distinct sites. Biphasicity was not observed in ITC experiments that analyzed the binding of Ac-CoA to a C-terminal construct of SePat encompassing the predicted core acetyltransferase domain. Subsequent analytical gel filtration chromatography studies showed that in the presence of Ac-CoA, SePat oligomerized to a tetrameric form, whereas in the absence of Ac-CoA, SePat behaved as a monomer. The positive modulation of SePat activity by Ac-CoA, a product of the Acs enzyme that also serves as a substrate for SePat-dependent acetylation, is likely a layer of metabolic control

The PlcR Virulence Regulon of Bacillus cereus

PlcR is a Bacillus cereus transcriptional regulator, which activates gene expression by binding to a nucleotidic sequence called the ‘PlcR box’. To build a list of all genes included in the PlcR regulon, a consensus sequence was identified by directed mutagenesis. The reference strain ATCC14579 sequenced genome was searched for occurrences of this consensus sequence to produce a virtual regulon. PlcR control of these genes was confirmed by comparing gene expression in the reference strain and its isogenic Δ-plcR strain using DNA microarrays, lacZ fusions and proteomics methods. The resulting list included 45 genes controlled by 28 PlcR boxes. Forty of the PlcR controlled proteins were exported, of which 22 were secreted in the extracellular medium and 18 were bound or attached to cell wall structures (membrane or peptidoglycan layer). The functions of these proteins were related to food supply (phospholipases, proteases, toxins), cell protection (bacteriocins, toxins, transporters, cell wall biogenesis) and environment-sensing (two-component sensors, chemotaxis proteins, GGDEF family regulators). Four genes coded for cytoplasmic regulators. The PlcR regulon appears to integrate a large range of environmental signals, including food deprivation and self cell-density, and regulate the transcription of genes designed to overcome obstacles that hinder B. cereus growth within the host: food supply, host barriers, host immune defenses, and competition with other bacterial species. PlcR appears to be a key component in the efficient adaptation of B. cereus to its host environment

Crystal Structures of Malonyl-Coenzyme A Decarboxylase Provide Insights into Its Catalytic Mechanism and Disease-Causing Mutations

Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design

Properties and identification of antibiotic drug targets

<p>Abstract</p> <p>Background</p> <p>We analysed 48 non-redundant antibiotic target proteins from all bacteria, 22 antibiotic target proteins from <it>E. coli </it>only and 4243 non-drug targets from <it>E. coli </it>to identify differences in their properties and to predict new potential drug targets.</p> <p>Results</p> <p>When compared to non-targets, bacterial antibiotic targets tend to be long, have high β-sheet and low α-helix contents, are polar, are found in the cytoplasm rather than in membranes, and are usually enzymes, with ligases particularly favoured. Sequence features were used to build a support vector machine model for <it>E. coli </it>proteins, allowing the assignment of any sequence to the drug target or non-target classes, with an accuracy in the training set of 94%. We identified 319 proteins (7%) in the non-target set that have target-like properties, many of which have unknown function. 63 of these proteins have significant and undesirable similarity to a human protein, leaving 256 target like proteins that are not present in humans.</p> <p>Conclusions</p> <p>We suggest that antibiotic discovery programs would be more likely to succeed if new targets are chosen from this set of target like proteins or their homologues. In particular, 64 are essential genes where the cell is not able to recover from a random insertion disruption.</p