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Isoniazid Pharmacokinetics-Pharmacodynamics in an Aerosol Infection Model of Tuberculosis. Ramesh Jayaram, 2004.Limited data exist on the pharmacokinetic-pharmacodynamic (PK-PD) parameters of the bactericidal activities of the available antimycobacterial drugs . We report on the PK-PD relationships for isoniazid . Isoniazid exhibited concentration (C)-dependent killing of Mycobacterium tuberculosis H37Rv in vitro, with a maximum reduction of 4 log10 CFU/ml . In these studies, 50% of the maximum effect was achieved at a C/MIC ratio of 0.5, and the maximum effect did not increase with exposure times of up to 21 days . Conversely, isoniazid produced less than a 0.5-log10 CFU/ml reduction in two different intracellular infection models (J774A.1 murine macrophages and whole human blood) . In a murine model of aerosol infection, isoniazid therapy for 6 days produced a reduction of 1.4 log10 CFU/lung . Dose fractionation studies demonstrated that the 24-h area under the concentration-time curve/MIC (r2 = 0.83) correlated best with the bactericidal efficacy, followed by the maximum concentration of drug in serum/MIC (r2 = 0.73) . Specific Response of a Novel and Abundant Lactobacillus amylovorus-Like Phylotype to Dietary Prebiotics in the Guts of Weaning Piglets. Sergey R. Konstantinov, 2004.Using 16S rRNA gene-based approaches, we analyzed the responses of ileal and colonic bacterial communities of weaning piglets to dietary addition of four fermentable carbohydrates (inulin, lactulose, wheat starch, and sugar beet pulp) . An enriched diet and a control diet lacking these fermentable carbohydrates were fed to piglets for 4 days (n = 48), and 10 days (n = 48), and the lumen-associated microbiota were compared using denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16S rRNA genes . Bacterial diversities in the ileal and colonic samples were measured by assessing the number of DGGE bands and the Shannon index of diversity . A higher number of DGGE bands in the colon (24.2 ± 5.5) than in the ileum (9.7 ± 4.2) was observed in all samples . In addition, significantly higher diversity, as measured by DGGE fingerprint analysis, was detected in the colonic microbial community of weaning piglets fed the fermentable-carbohydrate-enriched diet for 10 days than in the control . Selected samples from the ileal and colonic lumens were also investigated using fluorescent in situ hybridization (FISH) and cloning and sequencing of the 16S rRNA gene . This revealed a prevalence of Lactobacillus reuteri in the ileum and Lactobacillus amylovorus-like populations in the ileum and the colon in the piglets fed with fermentable carbohydrates . Newly developed oligonucleotide probes targeting these phylotypes allowed their rapid detection and quantification in the ileum and colon by FISH . The results indicate that addition of fermentable carbohydrates supports the growth of specific lactobacilli in the ilea and colons of weaning piglets . Unique Presence of a Manganese Catalase in a Hyperthermophilic Archaeon, Pyrobaculum calidifontis VA1. Taku Amo, 2002.We had previously isolated a facultatively anaerobic hyperthermophilic archaeon, Pyrobaculum calidifontis strain VA1 . Here, we found that strain VA1, when grown under aerobic conditions, harbors high catalase activity . The catalase was purified 91-fold from crude extracts and displayed a specific activity of 23,500 U/mg at 70°C . The enzyme exhibited a Km value of 170 mM toward H2O2 and a kcat value of 2.9 x 104 s-1·subunit-1 at 25°C . Gel filtration chromatography indicated that the enzyme was a homotetramer with a subunit molecular mass of 33,450 Da . The purified catalase did not display the Soret band, which is an absorption band particular to heme enzymes . In contrast to typical heme catalases, the catalase was not strongly inhibited by sodium azide . Furthermore, with plasma emission spectroscopy, we found that the catalase did not contain iron but instead contained manganese . Our biochemical results indicated that the purified catalase was not a heme catalase but a manganese (nonheme) catalase, the first example in archaea . Intracellular catalase activity decreased when cells were grown anaerobically, while under aerobic conditions, an increase in activity was observed with the removal of thiosulfate from the medium, or addition of manganese . Based on the N-terminal amino acid sequence of the purified protein, we cloned and sequenced the catalase gene (katPc) . The deduced amino acid sequence showed similarity with that of the manganese catalase from a thermophilic bacterium, Thermus sp . YS 8-13 . Interestingly, in the complete archaeal genome sequences, no open reading frame has been assigned as a manganese catalase gene . Moreover, a homology search with the sequence of katPc revealed that no orthologue genes were present on the archaeal genomes, including those from the "aerobic" (hyper)thermophilic archaea Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii . Therefore, KatPc can be considered a rare example of a manganese catalase from archaea . Genetics of Streptococci, Lactococci, and Enterococci: Review of the Sixth International Conference. Janet Yother, 2002. Interdomain Linkers of Homologous Response Regulators Determine Their Mechanism of Action. Don Walthers, 2003.OmpR and PhoB are response regulators that contain an N-terminal phosphorylation domain and a C-terminal DNA binding effector domain connected by a flexible interdomain linker . Phosphorylation of the N terminus results in an increase in affinity for specific DNA and the subsequent regulation of gene expression . Despite their sequence and structural similarity, OmpR and PhoB employ different mechanisms to regulate their effector domains . Phosphorylation of OmpR in the N terminus stimulates the DNA binding affinity of the C terminus, whereas phosphorylation of the PhoB N terminus relieves inhibition of the C terminus, enabling it to bind to DNA . Chimeras between OmpR and PhoB containing either interdomain linker were constructed to explore the basis of the differences in their activation mechanisms . Our results indicate that effector domain regulation by either N terminus requires its cognate interdomain linker . In addition, our findings suggest that the isolated C terminus of OmpR is not sufficient for a productive interaction with RNA polymerase . Organization and Expression of the Polynucleotide Phosphorylase Gene (pnp) of Streptomyces: Processing of pnp Transcripts in Streptomyces antibioticus. Patricia Bralley, 2004.We have examined the expression of pnp encoding the 3'-5'-exoribonuclease, polynucleotide phosphorylase, in Streptomyces antibioticus . We show that the rpsO-pnp operon is transcribed from at least two promoters, the first producing a readthrough transcript that includes both pnp and the gene for ribosomal protein S15 (rpsO) and a second, Ppnp, located in the rpsO-pnp intergenic region . Unlike the situation in Escherichia coli, where observation of the readthrough transcript requires mutants lacking RNase III, we detect readthrough transcripts in wild-type S . antibioticus mycelia . The Ppnp transcriptional start point was mapped by primer extension and confirmed by RNA ligase-mediated reverse transcription-PCR, a technique which discriminates between 5' ends created by transcription initiation and those produced by posttranscriptional processing . Promoter probe analysis demonstrated the presence of a functional promoter in the intergenic region . The Ppnp sequence is similar to a group of promoters recognized by the extracytoplasmic function sigma factors, sigma-R and sigma-E . We note a number of other differences in rspO-pnp structure and function between S . antibioticus and E . coli . In E . coli, pnp autoregulation and cold shock adaptation are dependent upon RNase III cleavage of an rpsO-pnp intergenic hairpin . Computer modeling of the secondary structure of the S . antibioticus readthrough transcript predicts a stem-loop structure analogous to that in E . coli . However, our analysis suggests that while the readthrough transcript observed in S . antibioticus may be processed by an RNase III-like activity, transcripts originating from Ppnp are not . Furthermore, the S . antibioticus rpsO-pnp intergenic region contains two open reading frames . The larger of these, orfA, may be a pseudogene . The smaller open reading frame, orfX, also observed in Streptomyces coelicolor and Streptomyces avermitilis, may be translationally coupled to pnp and the gene downstream from pnp, a putative protease .
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