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Novel Chemical Class of pUL97 Protein Kinase-Specific Inhibitors with Strong Anticytomegaloviral Activity. Thomas Herget, 2004.Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns . The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication . Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication . We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC50s) between 30 and 170 nM . Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency . The IC50s of these three quinazoline compounds (2.4 ± 0.4, 3.4 ± 0.6, and 3.9 ± 1.1 µM, respectively) were in the range of the IC50 of ganciclovir (1.2 ± 0.2 µM), as determined by the HCMV green fluorescent protein-based antiviral assay . Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir- and cidofovir-resistant virus variants . Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed . The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells . Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function . In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption . Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity . Oxidation of Methyl tert-Butyl Ether by Alkane Hydroxylase in Dicyclopropylketone-Induced and n-Octane-Grown Pseudomonas putida GPo1. Christy A. Smith, 2004.The alkane hydroxylase enzyme system in Pseudomonas putida GPo1 has previously been reported to be unreactive toward the gasoline oxygenate methyl tert-butyl ether (MTBE) . We have reexamined this finding by using cells of strain GPo1 grown in rich medium containing dicyclopropylketone (DCPK), a potent gratuitous inducer of alkane hydroxylase activity . Cells grown with DCPK oxidized MTBE and generated stoichiometric quantities of tert-butyl alcohol (TBA) . Cells grown in the presence of DCPK also oxidized tert-amyl methyl ether but did not appear to oxidize either TBA, ethyl tert-butyl ether, or tert-amyl alcohol . Evidence linking MTBE oxidation to alkane hydroxylase activity was obtained through several approaches . First, no TBA production from MTBE was observed with cells of strain GPo1 grown on rich medium without DCPK . Second, no TBA production from MTBE was observed in DCPK-treated cells of P . putida GPo12, a strain that lacks the alkane-hydroxylase-encoding OCT plasmid . Third, all n-alkanes that support the growth of strain GPo1 inhibited MTBE oxidation by DCPK-treated cells . Fourth, two non-growth-supporting n-alkanes (propane and n-butane) inhibited MTBE oxidation in a saturable, concentration-dependent process . Fifth, 1,7-octadiyne, a putative mechanism-based inactivator of alkane hydroxylase, fully inhibited TBA production from MTBE . Sixth, MTBE-oxidizing activity was also observed in n-octane-grown cells . Kinetic studies with strain GPo1 grown on n-octane or rich medium with DCPK suggest that MTBE-oxidizing activity may have previously gone undetected in n-octane-grown cells because of the unusually high Ks value (20 to 40 mM) for MTBE . Gene Cloning, Purification, and Characterization of a Phosphodiesterase from Delftia acidovorans. Sundiep K. Tehara, 2003.A novel phosphodiesterase (PdeA) was purified from Delftia acidovorans, the gene encoding the enzyme was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to apparent homogeneity and characterized . PdeA is an 85-kDa trimer that exhibits maximal activity at 65°C and pH 10 even though it was isolated from a mesophilic bacterium . Although PdeA exhibited both mono- and diesterase activity, it was most active on the phosphodiester bis(p-nitrophenyl)phosphate with a Km of 2.9 ± 0.1 mM and a kcat of 879 ± 73 min-1 . The enzyme showed sequence similarity to cyclic AMP (cAMP) phosphodiesterase and cyclic nucleotide phosphodiesterases and exhibited activity on cAMP in vivo when the gene was expressed in E . coli . The IS1071 transposon insertion sequence was found downstream of pdeA . Fluorescent Heteroduplex Assay for Monitoring Bacillus anthracis and Close Relatives in Environmental Samples. Lori Merrill, 2003.A fluorescent heteroduplex method was developed to assess the presence of 16S rRNA gene (rDNA) sequences from Bacillus anthracis and close relatives in PCR-amplified 16S rDNA sequence mixtures from environmental samples . The method uses a single-stranded, fluorescent DNA probe, 464 nucleotides in length, derived from a B . anthracis 16S rRNA gene . The probe contains a unique, engineered deletion such that all probe-target duplexes are heteroduplexes with an unpaired G at position 343 (  G343) . Heteroduplex profiles of sequences
 85% similar to the probe were produced using an ABI 377 sequencer in less than 3 h . The method divides strains of the Bacillus cereus-Bacillus thuringiensis-B . anthracis group into two subgroups . Each subgroup is defined by a specific 16S rRNA gene sequence type . Sequence type A, containing one mismatch with the probe, occurs in B . anthracis and a small number of closely related clonal lineages represented mostly by food-borne pathogenic isolates of B . cereus and B . thuringiensis . Sequence type B, containing two mismatches with the probe, is found in the majority of B . cereus and B . thuringiensis strains examined to date . Sequence types A and B, when hybridized to the probe, generate two easily differentiated heteroduplexes . Thus, from heteroduplex profiles, the presence of B . cereus-B . thuringiensis-B . anthracis subgroups in environmental samples can be inferred unambiguously . The results show that fluorescent heteroduplex analysis is an effective profiling technique for detection and differentiation of sequences representing small phylogenetic or functional groups in environmental samples .
Patterns of Community Change among Ammonia Oxidizers in Meadow Soils upon Long-Term Incubation at Different Temperatures. Sharon Avrahami, 2003.The effect of temperature on the community structure of ammonia-oxidizing bacteria was investigated in three different meadow soils . Two of the soils (OMS and GMS) were acidic (pH 5.0 to 5.8) and from sites in Germany with low annual mean temperature (about 10°C), while KMS soil was slightly alkaline (pH 7.9) and from a site in Israel with a high annual mean temperature (about 22°C) . The soils were fertilized and incubated for up to 20 weeks in a moist state and as a buffered (pH 7) slurry amended with urea at different incubation temperatures (4 to 37°C) . OMS soil was also incubated with less fertilizer than the other soils . The community structure of ammonia oxidizers was analyzed before and after incubation by denaturing gradient gel electrophoresis (DGGE) of the amoA gene, which codes for the  subunit of ammonia monooxygenase . All amoA gene sequences found belonged to the genus Nitrosospira . The analysis showed community change due to temperature both in moist soil and in the soil slurry . Two patterns of community change were observed . One pattern was a change between the different Nitrosospira clusters, which was observed in moist soil and slurry incubations of GMS and OMS . Nitrosospira AmoA cluster 1 was mainly detected below 30°C, while Nitrosospira cluster 4 was predominant at 25°C . Nitrosospira clusters 3a, 3b, and 9 dominated at 30°C . The second pattern, observed in KMS, showed a community shift predominantly within a single Nitrosospira cluster . The sequences of the individual DGGE bands that exhibited different trends with temperature belonged almost exclusively to Nitrosospira cluster 3a . We conclude that ammonia oxidizer populations are influenced by temperature . In addition, we confirmed previous observations that N fertilizer also influences the community structure of ammonia oxidizers . Thus, Nitrosospira cluster 1 was absent in OMS soil treated with less fertilizer, while Nitrosospira cluster 9 was only found in the sample given less fertilizer .
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