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In Vitro Activity of Expanded-Spectrum Pyridazinyl Oxime Ethers Related to Pirodavir: Novel Capsid-Binding Inhibitors with Potent Antipicornavirus Activity. D. L. Barnard, 2004.Picornaviruses (PV) include human rhinovirus (HRV), the primary cause of the common cold, and the enteroviruses (EV), which cause serious diseases such as poliomyelitis, meningoencephalitis, and systemic neonatal disease . Although no compounds for PV infections have been approved in the United States, pirodavir was one of the most promising capsid-binding compounds to show efficacy in human clinical trials for chemoprophylaxis of the common cold . Susceptibility to hydrolysis precluded its use as an oral agent . We have developed orally bioavailable pyridazinyl oxime ethers that are as potent as pirodavir . Compounds BTA39 and BTA188 inhibited a total of 56 HRV laboratory strains and three clinical isolates as determined by neutral red uptake assay . At concentrations of <100 nM, BTA39 inhibited 69% of the HRV serotypes and isolates evaluated, BTA188 inhibited 75%, and pirodavir inhibited 59% of the serotypes and isolates . The 50% inhibitory concentrations (IC50s) for the two compounds ranged from 0.5 nM to 6,701 nM . The compounds also inhibited EV, including coxsackie A and B viruses (IC50 = 773 to 3,608 nM) and echoviruses (IC50 = 193 to 5,155 nM) . BTA39 only inhibited poliovirus strain WM-1 at 204 nM, and BTA188 only inhibited poliovirus strain Chat at 82 nM . EV 71 was inhibited by BTA39 and BTA188, with IC50s of 1 and 82 nM, respectively . Both compounds were relatively nontoxic in actively growing cells (50% cytotoxic doses,  4,588 nM) . These data suggest that these oxime ethers warrant further investigation as potential agents for treating selected PV infections .
Salmonella enterica Serovar Typhimurium Resistance to Bile: Identification and Characterization of the tolQRA Cluster. Angela M. Prouty, 2002.Salmonella enterica serovar Typhimurium is resistant to the action of bile salts, and resistance to bile is enhanced in strains in which the PhoP-PhoQ (PhoPQ) two-component regulatory system has been activated . To identify genes necessary for bile resistance, MudJ transposon mutagenesis was performed on a strain containing a phoP mutation that results in constitutive expression of PhoP-activated genes . After screening >10,000 mutants for the loss of growth on Luria-Bertani broth-bile plates, 14 bile-sensitive mutants were identified . Of these 14 mutants, 3 were found to retain the bile sensitivity phenotype upon P22 transduction, to possess wild-type growth characteristics, and to contain a smooth lipopolysaccharide . Southern hybridization experiments showed that all three strains contained unique insertions . DNA sequencing of the transposon-chromosomal-DNA fusion junctions of these strains showed all to be linked to the putative Salmonella orf1-tolQRA operon, with insertions in tolQ, orf1, and a gene upstream of the orf1-tolQRA operon not previously associated with Tol function (orfX) . Through the use of transcriptional fusions, none of the putative tol (or tol-associated) genes were shown to be regulated by PhoPQ, bile, or the RcsC-RcsB two-component system; however, all of the genes (orfX, orf1, tolQRA) are predicted to be cotranscribed . This is the first identification of Salmonella serovar Typhimurium Tol homologs and the first demonstration of their role in bile resistance in this organism . In addition, the observed regulation, operon arrangement, and phenotypes associated with these tol genes demonstrate significant differences from their Escherichia coli homologs . The Escherichia coli lipB Gene Encodes Lipoyl (Octanoyl)-Acyl Carrier Protein:Protein Transferase. Sean W. Jordan, 2003.In an earlier study (S . W . Jordan and J . E . Cronan, Jr., J . Biol . Chem . 272:17903-17906, 1997) we reported a new enzyme, lipoyl-[acyl carrier protein]-protein N-lipoyltransferase, in Escherichia coli and mitochondria that transfers lipoic acid from lipoyl-acyl carrier protein to the lipoyl domains of pyruvate dehydrogenase . It was also shown that E . coli lipB mutants lack this enzyme activity, a finding consistent with lipB being the gene that encoded the lipoyltransferase . However, it remained possible that lipB encoded a positive regulator required for lipoyltransferase expression or action . We now report genetic and biochemical evidence demonstrating that lipB encodes the lipoyltransferase . A lipB temperature-sensitive mutant was shown to produce a thermolabile lipoyltransferase and a tagged version of the lipB-encoded protein was purified to homogeneity and shown to catalyze the transfer of either lipoic acid or octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase . In the course of these experiments the ATG initiation codon commonly assigned to lipB genes in genomic databases was shown to produce a nonfunctional E . coli LipB protein, whereas initiation at an upstream TTG codon gave a stable and enzymatically active protein . Prior genetic results (T . W . Morris, K . E . Reed, and J . E . Cronan, Jr., J . Bacteriol . 177:1-10, 1995) suggested that lipoate protein ligase (LplA) could also utilize (albeit poorly) acyl carrier protein substrates in addition to its normal substrates lipoic acid plus ATP . We have detected a very slow LplA-catalyzed transfer of lipoic acid and octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase . A nonhydrolyzable lipoyl-AMP analogue was found to competitively inhibit both ACP-dependent and ATP-dependent reactions of LplA, suggesting that the same active site catalyzes two chemically diverse reactions . Temporal Stability and Biodiversity of Two Complex Antilisterial Cheese-Ripening Microbial Consortia. Ariel Maoz, 2003.The temporal stability and diversity of bacterial species composition as well as the antilisterial potential of two different, complex, and undefined microbial consortia from red-smear soft cheeses were investigated . Samples were collected twice, at 6-month intervals, from each of two food producers, and a total of 400 bacterial isolates were identified by Fourier-transform infrared spectroscopy and 16S ribosomal DNA sequence analysis . Coryneform bacteria represented the majority of the isolates, with certain species being predominant . In addition, Marinolactobacillus psychrotolerans, Halomonas venusta, Halomonas variabilis, Halomonas sp . (106 to 107 CFU per g of smear), and an unknown, gram-positive bacterium (107 to 108 CFU per g of smear) are described for the first time in such a consortium . The species composition of one consortium was quite stable over 6 months, but the other consortium revealed less diversity of coryneform species as well as less stability . While the first consortium had a stable, extraordinarily high antilisterial potential in situ, the antilisterial activity of the second consortium was lower and decreased with time . The cause for the antilisterial activity of the two consortia remained unknown but is not due to the secretion of soluble, inhibitory substances by the individual components of the consortium . Our data indicate that the stability over time and a potential antilisterial activity are individual characteristics of the ripening consortia which can be monitored and used for safe food production without artificial preservatives .
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