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Biotech / Medical : Essential Therapeutics (ETRX) formerly Microcide (MCDE -- Ignore unavailable to you. Want to Upgrade?

To: SemiBull who wrote (205)	8/20/1999 6:46:00 PM
From: scaram(o)uche	Read Replies (2) \| Respond to of 415

scouring around, getting ready for ICAAC, and ran across this.... J Bacteriol 1999 Jun;181(12):3666-73 Role in cell permeability of an essential two-component system in Staphylococcus aureus. Martin PK, Li T, Sun D, Biek DP, Schmid MB Microcide Pharmaceuticals, Inc., Mountain View, California 94043, USA. martin@microcide.com A temperature-sensitive lethal mutant of Staphylococcus aureus was found to harbor a mutation in the uncharacterized two-component histidine kinase (HK)-response regulator (RR) pair encoded by yycFG; orthologues of yycFG could be identified in the genomes of Bacillus subtilis and other gram-positive bacteria. Sequence analysis of the mutant revealed a point mutation resulting in a nonconservative change (Glu to Lys) in the regulator domain of the RR at position 63. To confirm that this signal transduction system was essential, a disrupted copy of either the RR (yycF) or the HK (yycG) was constructed with a set of suicide vectors and used to generate tandem duplications in the chromosome. Resolution of the duplications, leaving an insertion in either the yycF or the yycG coding region, was achieved only in the presence of an additional wild-type copy of the two open reading frames. Phenotypic characterization of the conditional lethal mutant showed that at permissive growth conditions, the mutant was hypersusceptible to macrolide and lincosamide antibiotics, even in the presence of the ermB resistance determinant. Other mutant phenotypes, including hypersensitivity to unsaturated long-chain fatty acids and suppression of the conditional lethal phenotype by high sucrose and NaCl concentrations, suggest that the role of the two-component system includes the proper regulation of bacterial cell wall or membrane composition. The effects of this point mutation are strongly bactericidal at the nonpermissive temperature, indicating that this pathway provides an excellent target for the identification of novel antibiotics.

To: SemiBull who wrote (205)	8/20/1999 6:48:00 PM
From: scaram(o)uche	Respond to of 415

another recent publication....... Antimicrob Agents Chemother 1999 Jun;43(6):1340-6 Use of a genetic approach to evaluate the consequences of inhibition of efflux pumps in Pseudomonas aeruginosa. Lomovskaya O, Lee A, Hoshino K, Ishida H, Mistry A, Warren MS, Boyer E, Chamberland S, Lee VJ Microcide Pharmaceuticals Inc., Mountain View, California 94043, USA. olga@microcide.com Drug efflux pumps in Pseudomonas aeruginosa were evaluated as potential targets for antibacterial therapy. The potential effects of pump inhibition on susceptibility to fluoroquinolone antibiotics were studied with isogenic strains that overexpress or lack individual efflux pumps and that have various combinations of efflux- and target-mediated mutations. Deletions in three efflux pump operons were constructed. As expected, deletion of the MexAB-OprM efflux pump decreased resistance to fluoroquinolones in the wild-type P. aeruginosa (16-fold reduction for levofloxacin [LVX]) or in the strain that overexpressed mexAB-oprM operon (64-fold reduction for LVX). In addition to that, resistance to LVX was significantly reduced even for the strains carrying target mutations (64-fold for strains for which LVX MICs were >4 microg/ml). We also studied the frequencies of emergence of LVX-resistant variants from different deletion mutants and the wild-type strain. Deletion of individual pumps or pairs of the pumps did not significantly affect the frequency of emergence of resistant variants (at 4x the MIC for the wild-type strain) compared to that for the wild type (10(-6) to 10(-7)). In the case of the strain with a triple deletion, the frequency of spontaneous mutants was undetectable (<10(-11)). In summary, inhibition of drug efflux pumps would (i) significantly decrease the level of intrinsic resistance, (ii) reverse acquired resistance, and (iii) result in a decreased frequency of emergence of P. aeruginosa strains highly resistant to fluoroquinolones in clinical settings.

To: SemiBull who wrote (205)	8/20/1999 6:50:00 PM
From: scaram(o)uche	Respond to of 415

and a new one from Iconix...... J Virol 1999 Jul;73(7):5663-70 A recombinant human cytomegalovirus with a large deletion in UL97 has a severe replication deficiency. Prichard MN, Gao N, Jairath S, Mulamba G, Krosky P, Coen DM, Parker BO, Pari GS Iconix Pharmaceuticals Inc., 850 Maude Ave., Mountain View, CA 94043, USA. mprichard@iconixpharm.com Human cytomegalovirus encodes a protein kinase (UL97) that confers sensitivity to ganciclovir by phosphorylating it to the monophosphate. The function of this unusual kinase in viral replication is unknown. We constructed two independent isolates of a recombinant virus, RCDelta97, that contain large deletions in this gene and carry a 4.8-kb insertion containing a selectable genetic marker. These mutant viruses were isolated by using a population of primary cells (HEL97) that express this gene from integrated copies of a defective retroviral vector. The recombinant viruses were severely impaired in their ability to replicate in primary fibroblasts, attaining virus titers that were 2 to 3 orders of magnitude lower than those produced by the parent virus. Despite the severe replication deficit, both of these viruses retained the ability to form small, slowly growing plaques in primary fibroblasts, demonstrating that UL97 is not absolutely essential for replication in cell culture. The replication deficit was relieved when UL97 was provided in trans in the complementing cell line, showing that the phenotype was due to a deficiency in UL97. Thus, the UL97 gene product plays a very important role in viral replication in tissue culture and may be a good target for antiviral chemotherapy.