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Antibody Mapping of the Linear Epitopes of CMY-2 and SHV-1 ß-Lactamases. Andrea M. Hujer, 2004.Knowledge of the amino acids that define recognition of anti-ß-lactamase antibodies is critical to the interpretation of sensitivity and specificity of these antibodies when they are used in a clinical or research setting . To this end, we mapped the epitopes of the CMY-2 and SHV-1 ß-lactamases by using the SPOT synthesis method . Eight linear epitopes in SHV-1 and seven linear epitopes in CMY-2 were identified by using anti-SHV-1 and anti-CMY-2 polyclonal antibodies, respectively . The epitopes of SHV-1 were mapped to amino acids at the Ambler positions ABL 28 to 38, 42 to 54, 88 to 100, 102 to 114, 170 to 182, 186 to 194, 202 to 210, and 276 to 288 . In the epitope spanning amino acids 102 to 114, alanine and X-Scan analysis demonstrated that D104, Y105, P107, and S109 are essential residues for antibody recognition . In the epitope containing amino acids 170 to 182, N170, L173, P174, G175, and D176 were immunodominant . In CMY-2 ß-lactamase, amino acids 4 to 16, 70 to 79, 211 to 223, 274 to 286, 289 to 298, 322 to 334, and 343 to 358 of the mature enzyme defined the major linear epitopes . A detailed analysis of the recognition sites that are located in an area analogous to the omega loop of class A ß-lactamases (V211 to V223) showed that the amino acids Q215 to E219 are important in antibody binding . Incubation of CMY-2 ß-lactamase with a 10-fold molar excess of anti-CMY-2 antibody for 60 min resulted in greater than 80% inhibition of nitrocefin hydrolysis . A 10-fold molar excess of anti-SHV-1 antibody reduced the activity of SHV-1 by 69% . Analysis of the CMY-2 and SHV-1 structures suggest that this reduction of hydrolytic activity may be due in part to the direct binding of antibodies to the omega loop, thereby hindering access of substrate to the active site . Screening and Characterization of Mutations in Isoniazid-Resistant Mycobacterium tuberculosis Isolates Obtained in Brazil. Rosilene Fressatti Cardoso, 2004.We investigated mutations in the genes katG, inhA (regulatory and structural regions), and kasA and the oxyR-ahpC intergenic region of 97 isoniazid (INH)-resistant and 60 INH-susceptible Mycobacterium tuberculosis isolates obtained in two states in Brazil: São Paulo and Paraná . PCR-single-strand conformational polymorphism (PCR-SSCP) was evaluated for screening mutations in regions of prevalence, including codons 315 and 463 of katG, the regulatory region and codons 16 and 94 of inhA, kasA, and the oxyR-ahpC intergenic region . DNA sequencing of PCR amplicons was performed for all isolates with altered PCR-SSCP profiles . Mutations in katG were found in 83 (85.6%) of the 97 INH-resistant isolates, including mutations in codon 315 that occurred in 60 (61.9%) of the INH-resistant isolates and 23 previously unreported katG mutations . Mutations in the inhA promoter region occurred in 25 (25.8%) of the INH-resistant isolates; 6.2% of the isolates had inhA structural gene mutations, and 10.3% had mutations in the oxyR-ahpC intergenic region (one, nucleotide –48, previously unreported) . Polymorphisms in the kasA gene occurred in both INH-resistant and INH-susceptible isolates . The most frequent polymorphism encoded a G269A substitution . Although KatG315 substitutions are predominant, novel mutations also appear to be responsible for INH resistance in the two states in Brazil . Since ca . 90.7% of the INH-resistant isolates had mutations identified by SSCP electrophoresis, this method may be a useful genotypic screen for INH resistance . Mutations in the CCGTTCACA DnaA Box of Mycobacterium tuberculosis oriC That Abolish Replication of oriC Plasmids Are Tolerated on the Chromosome. Jaroslaw Dziadek, 2002.The origin of replication (oriC) region in some clinical strains of Mycobacterium tuberculosis is a hot spot for IS6110 elements . To understand how clinical strains with insertions in oriC can replicate their DNA, we characterized the oriC regions of some clinical strains . Using a plasmid-based oriC-dependent replication assay, we showed that IS6110 insertions that disrupted the DnaA box sequence CCGTTCACA abolished oriC activity in M . tuberculosis . Furthermore, by using a surface plasmon resonance technique we showed that purified M . tuberculosis DnaA protein binds native but not mutant DnaA box sequence, suggesting that stable interactions of the DnaA protein with the CCGTTCACA DnaA box are crucial for replication of oriC plasmids in vivo . Replacement by homologous recombination of the CCGTTCACA DnaA box sequence of the laboratory strain M . tuberculosis H37Ra with a mutant sequence did not result in nonviability . Together, these results suggest that M . tuberculosis strains have evolved mechanisms to tolerate mutations in the oriC region and that functional requirements for M . tuberculosis oriC replication are different for chromosomes and plasmids . Interaction between the H2 Sensor HupUV and the Histidine Kinase HupT Controls HupSL Hydrogenase Synthesis in Rhodobacter capsulatus. Sylvie Elsen, 2003.The photosynthetic bacterium Rhodobacter capsulatus contains two [NiFe]hydrogenases: an energy-generating hydrogenase, HupSL, and a regulatory hydrogenase, HupUV . The synthesis of HupSL is specifically activated by H2 through a signal transduction cascade comprising three proteins: the H2-sensing HupUV protein, the histidine kinase HupT, and the transcriptional regulator HupR . Whereas a phosphotransfer between HupT and HupR was previously demonstrated, interaction between HupUV and HupT was only hypothesized based on in vivo analyses of mutant phenotypes . To visualize the in vitro interaction between HupUV and HupT proteins, a six-His (His6)-HupU fusion protein and the HupV protein were coproduced by using a homologous expression system . The two proteins copurified as a His6-HupUHupV complex present in dimeric and tetrameric forms, both of which had H2 uptake activity . We demonstrated that HupT and HupUV interact and form stable complexes that could be separated on a native gel . Interaction was also monitored with surface plasmon resonance technology and was shown to be insensitive to salt concentration and pH changes, suggesting that the interactions involve hydrophobic residues . As expected, H2 affects the interaction between HupUV and HupT, leading to a weakening of the interaction, which is independent of the phosphate status of HupT . Several forms of HupT were tested for their ability to interact with HupUV and to complement hupT mutants . Strong interaction with HupUV was obtained with the isolated PAS domain of HupT and with inactive HupT mutated in the phosphorylable histidine residue, but only the wild-type HupT protein was able to restore normal H2 regulation .
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