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Mutations Conferring Resistance to a Potent Hepatitis C Virus Serine Protease Inhibitor In Vitro. Liangjun Lu, 2004.BILN 2061 is a novel, specific hepatitis C virus (HCV) NS3 serine protease inhibitor discovered by Boehringer Ingelheim that has shown potent activity against HCV replicons in tissue culture and is currently under clinical investigation for the treatment of HCV infection . The poor fidelity of the HCV RNA-dependent RNA polymerase will likely lead to the development of drug-resistant viruses in treated patients . The development of resistance to BILN 2061 was studied by the in vitro passage of HCV genotype 1b replicon cells in the presence of a fixed concentration of the drug . Three weeks posttreatment, four colonies were expanded for genotypic and phenotypic characterization . The 50% inhibitory concentrations of BILN 2061 for these colonies were 72- to 1,228-fold higher than that for the wild-type replicon . Sequencing of the individual colonies identified several mutations in the NS3 serine protease gene . Molecular clones containing the single amino acid substitution A156T, R155Q, or D168V resulted in 357-fold, 24-fold, and 144-fold reductions in susceptibility to BILN 2061, respectively, compared to the level of susceptibility shown by the wild-type replicon . Modeling studies indicate that all three of these residues are located in close proximity to the inhibitor binding site . These findings, in addition to the three-dimensional structure analysis of the NS3/NS4A serine protease inhibitor complex, provide a strategic guide for the development of next-generation inhibitors of HCV NS3/NS4A serine protease . Sequences near the Active Site in Chimeric Penicillin Binding Proteins 5 and 6 Affect Uniform Morphology of Escherichia coli. Anindya S. Ghosh, 2003.Penicillin binding protein (PBP) 5, a DD-carboxypeptidase that removes the terminal D-alanine from peptide side chains of peptidoglycan, plays an important role in creating and maintaining the uniform cell shape of Escherichia coli . PBP 6, a highly similar homologue, cannot substitute for PBP 5 in this respect . Previously, we localized the shape-maintaining characteristics of PBP 5 to the globular domain that contains the active site (domain I), where PBPs 5 and 6 share substantial identity . To identify the specific segment of domain I responsible for shape control, we created a set of hybrids and determined which ones complemented the aberrant morphology of a misshapen PBP mutant, E . coli CS703-1 . Fusion proteins were constructed in which 47, 199 and 228 amino-terminal amino acids of one PBP were fused to the corresponding carboxy-terminal amino acids of the other . The morphological phenotype was reversed only by hybrid proteins containing PBP 5 residues 200 to 228, which are located next to the KTG motif of the active site . Because residues 220 to 228 were identical in these proteins, the morphological effect was determined by alterations in amino acids 200 to 219 . To confirm the importance of this segment, we constructed mosaic proteins in which these 20 amino acids were grafted from PBP 5 into PBP 6 and vice versa . The PBP 6/5/6 mosaic complemented the aberrant morphology of CS703-1, whereas PBP 5/6/5 did not . Site-directed mutagenesis demonstrated that the Asp218 and Lys219 residues were important for shape maintenance by these mosaic PBPs, but the same mutations in wild-type PBP 5 did not eliminate its shape-promoting activity . Homologous enzymes from five other bacteria also complemented the phenotype of CS703-1 . The overall conclusion is that creation of a bacterial cell of regular diameter and uniform contour apparently depends primarily on a slight alteration of the enzymatic activity or substrate accessibility at the active site of E . coli PBP 5 . Ubiquity and Persistence of Escherichia coli in a Midwestern Coastal Stream. Muruleedhara Byappanahalli, 2003.Dunes Creek, a small Lake Michigan coastal stream that drains sandy aquifers and wetlands of Indiana Dunes, has chronically elevated Escherichia coli levels along the bathing beach near its outfall . This study sought to understand the sources of E . coli in Dunes Creek's central branch . A systematic survey of random and fixed sampling points of water and sediment was conducted over 3 years . E . coli concentrations in Dunes Creek and beach water were significantly correlated . Weekly monitoring at 14 stations during 1999 and 2000 indicated chronic loading of E . coli throughout the stream . Significant correlations between E . coli numbers in stream water and stream sediment, submerged sediment and margin, and margin and 1 m from shore were found . Median E . coli counts were highest in stream sediments, followed by bank sediments, sediments along spring margins, stream water, and isolated pools; in forest soils, E . coli counts were more variable and relatively lower . Sediment moisture was significantly correlated with E . coli counts . Direct fecal input inadequately explains the widespread and consistent occurrence of E . coli in the Dunes Creek watershed; long-term survival or multiplication or both seem likely . The authors conclude that (i) E . coli is ubiquitous and persistent throughout the Dunes Creek basin, (ii) E . coli occurrence and distribution in riparian sediments help account for the continuous loading of the bacteria in Dunes Creek, and (iii) ditching of the stream, increased drainage, and subsequent loss of wetlands may account for the chronically high E . coli levels observed .
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