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Steady-State Plasma and Intrapulmonary Pharmacokinetics and Pharmacodynamics of Cethromycin. John E. Conte Jr., 2004.The objective of this study was to determine the steady-state plasma and intrapulmonary pharmacokinetic parameters of orally administered cethromycin in healthy volunteers . The study design included administering 150 or 300 mg of cethromycin once daily to 25 or 35 healthy adult subjects, respectively, for a total of five doses . Standardized and timed bronchoalveolar lavage (BAL) was performed after the last dose . Blood was obtained for drug assay prior to the first and last dose, at multiple time points following the last dose, and at the time of BAL . Cethromycin was measured in plasma, BAL, and alveolar cell (AC) by using a combined high-performance liquid chromatography-mass spectrometric technique . Plasma, epithelial lining fluid (ELF), and AC pharmacokinetics were derived by noncompartmental methods . Cmax/90% minimum inhibitory concentration (MIC90) ratios, area under the concentration-time curve (AUC)/MIC90 ratios, intrapulmonary drug exposure ratios, and percent time above MIC90 during the dosing interval (%T > MIC90) were calculated for recently reported respiratory pathogens . The kinetics were nonlinear, i.e., not proportional to dose . In the 150-mg-dose group, the Cmax (mean ± standard deviations), AUC0-24, and half-life for plasma were 0.181 ± 0.084 µg/ml, 0.902 ± 0.469 µg · h/ml, and 4.85 ± 1.10 h, respectively; for ELF the values were 0.9 ± 0.2 µg/ml, 11.4 µg · h/ml, and 6.43 h, respectively; for AC the values were 12.7 ± 6.4 µg/ml, 160.8 µg · h/ml, and 10.0 h, respectively . In the 300-mg-dose group, the Cmax (mean ± standard deviations), AUC0-24, and half-life for plasma were 0.500 ± 0.168 µg/ml, 3.067 ± 1.205 µg · h/ml, and 4.94 ± 0.66 h, respectively; for ELF the values were 2.7 ± 2.0 µg/ml, 24.15 µg · h/ml, and 5.26 h, respectively; for AC the values were 55.4 ± 38.7 µg/ml, 636.2 µg · h/ml, and 11.6 h, respectively . We concluded that the Cmax/MIC90 ratios, AUC/MIC90 ratios, %T > MIC90 values, and extended plasma and intrapulmonary half-lives provide a pharmacokinetic rationale for once-daily administration and are favorable for the treatment of cethromycin-susceptible pulmonary infections . Polyhydroxyalkanoate (PHA) Accumulation in Sulfate-Reducing Bacteria and Identification of a Class III PHA Synthase (PhaEC) in Desulfococcus multivorans. Tran Hai, 2004.Seven strains of sulfate-reducing bacteria (SRB) were tested for the accumulation of polyhydroxyalkanoates (PHAs) . During growth with benzoate Desulfonema magnum accumulated large amounts of poly(3-hydroxybutyrate) [poly(3HB)] . Desulfosarcina variabilis (during growth with benzoate), Desulfobotulus sapovorans (during growth with caproate), and Desulfobacterium autotrophicum (during growth with caproate) accumulated poly(3HB) that accounted for 20 to 43% of cell dry matter . Desulfobotulus sapovorans and Desulfobacterium autotrophicum also synthesized copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyvalerate when valerate was used as the growth substrate . Desulfovibrio vulgaris and Desulfotalea psychrophila were the only SRB tested in which PHAs were not detected . When total DNA isolated from Desulfococcus multivorans and specific primers deduced from highly conserved regions of known PHA synthases (PhaC) were used, a PCR product homologous to the central region of class III PHA synthases was obtained . The complete pha locus of Desulfococcus multivorans was subsequently obtained by inverse PCR, and it contained adjacent phaEDm and phaCDm genes . PhaCDm and PhaEDm were composed of 371 and 306 amino acid residues and showed up to 49 or 23% amino acid identity to the corresponding subunits of other class III PHA synthases . Constructs of phaCDm alone (pBBRMCS-2::phaCDm) and of phaEDmCDm (pBBRMCS-2::phaEDmCDm) in various vectors were obtained and transferred to several strains of Escherichia coli, as well as to the PHA-negative mutants PHB–4 and GPp104 of Ralstonia eutropha and Pseudomonas putida, respectively . In cells of the recombinant strains harboring phaEDmCDm small but significant amounts (up to 1.7% of cell dry matter) of poly(3HB) and of PHA synthase activity (up to 1.5 U/mg protein) were detected . This indicated that the cloned genes encode functionally active proteins . Hybrid synthases consisting of PhaCDm and PhaE of Thiococcus pfennigii or Synechocystis sp . strain PCC 6308 were also constructed and were shown to be functionally active . A Repressor Protein, PhaR, Regulates Polyhydroxyalkanoate (PHA) Synthesis via Its Direct Interaction with PHA. Akira Maehara, 2002.Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis . Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression . In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation . Many TGC-rich sequences were found in upstream elements recognized by PhaR . PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules . Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation . These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA . However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins . An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB) . Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis . Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis . Requirement for IscS in Biosynthesis of All Thionucleosides in Escherichia coli. Charles T. Lauhon, 2002.Escherichia coli tRNA contains four naturally occurring nucleosides modified with sulfur . Cysteine is the intracellular sulfur source for each of these modified bases . We previously found that the iscS gene, a member of the nifS cysteine desulfurase gene family, is required for 4-thiouridine biosynthesis in E . coli . Since IscS does not bind tRNA, its role is the mobilization and distribution of sulfur to enzymes that catalyze the sulfur insertion steps . In addition to iscS, E . coli contains two other nifS homologs, csdA and csdB, each of which has cysteine desulfurase activity and could potentially donate sulfur for thionucleoside biosynthesis . Double csdA csdB and iscS csdA mutants were prepared or obtained, and all mutants were analyzed for thionucleoside content . It was found that unfractionated tRNA isolated from the iscS mutant strain contained <5% of the level of sulfur found in the parent strain . High-pressure liquid chromatography analysis of tRNA nuclease digests from the mutant strain grown in the presence of [35S]cysteine showed that only a small fraction of 2-thiocytidine was present, while the other thionucleosides were absent when cells were isolated during log phase . As expected, digests from the iscS mutant strain contained 6-N-dimethylallyl adenosine (i6A) in place of 6-N-dimethylallyl-2-methylthioadenosine and 5-methylaminomethyl uridine (mnm5U) instead of 5-methylaminomethyl-2-thiouridine . Prolonged growth of the iscS and iscS csdA mutant strains revealed a gradual increase in levels of 2-thiocytidine and 6-N-dimethylallyl-2-methylthioadenosine with extended incubation (>24 h), while the thiouridines remained absent . This may be due to a residual level of Fe-S cluster biosynthesis in iscS deletion strains . An overall scheme for thionucleoside biosynthesis in E . coli is discussed . Expression of the Secondary Sigma Factor  X in Streptococcus pyogenes Is Restricted at Two Levels.Jason A. Opdyke, 2003.Secondary RNA polymerase sigma factors in many bacteria are responsible for regulating a vast range of processes including virulence . A protein ( X) in the gram-positive human pathogen Streptococcus pyogenes (the group A Streptococcus or GAS) was recently shown to function in vitro as a secondary sigma factor . We report here the isolation of a mutant in which both sigX genes are inactivated, show that
X functions in GAS cells, and show that the amount of
X is controlled at two levels . Primer extension analysis indicates that sigX transcription is low in GAS cells grown in Todd-Hewitt yeast broth, and immunoblot assays with a
X-specific polyclonal antibody demonstrate that the protein does not accumulate in these cells . To increase the level of sigX transcription in GAS, we constructed a strain that constitutively expresses the sigX gene from a heterologous promoter . Expression of sigX from this promoter led to transcription of the
X-dependent cinA promoter in GAS cells . We found that expression of the sigX gene in a clpP mutant strain resulted in greater accumulation of
X protein, which resulted in higher levels of transcription from the
X-dependent promoters cinA, smf, and cglA . In addition, a clpP mutant containing sigX only at its wild-type loci on the chromosome generated more transcription from the
X-dependent cinA promoter than did the wild-type parental strain . Therefore,
X activity in GAS is limited by low-level transcription of the sigX structural genes and by clpP, which appears to negatively regulate
X accumulation .
Community Analysis of Ammonia and Nitrite Oxidizers during Start-Up of Nitritation Reactors. Konrad Egli, 2003.Partial nitrification of ammonium to nitrite under oxic conditions (nitritation) is a critical process for the effective use of alternative nitrogen removal technologies from wastewater . Here we investigated the conditions which promote establishment of a suitable microbial community for performing nitritation when starting from regular sewage sludge . Reactors were operated in duplicate under different conditions (pH, temperature, and dilution rate) and were fed with 50 mM ammonium either as synthetic medium or as sludge digester supernatant . In all cases, stable nitritation could be achieved within 10 to 20 days after inoculation . Quantitative in situ hybridization analysis with group-specific fluorescent rRNA-targeted oligonucleotides (FISH) in the different reactors showed that nitrite-oxidizing bacteria of the genus Nitrospira were only active directly after inoculation with sewage sludge (up to 4 days and detectable up to 10 days) . As demonstrated by quantitative FISH and restriction fragment length polymorphism (RFLP) analyses of the amoA gene (encoding the active-site subunit of the ammonium monooxygenase), the community of ammonia-oxidizing bacteria changed within the first 15 to 20 days from a more diverse set of populations consisting of members of the Nitrosomonas communis and Nitrosomonas oligotropha sublineages and the Nitrosomonas europaea-Nitrosomonas eutropha subgroup in the inoculated sludge to a smaller subset in the reactors . Reactors operated at 30°C and pH 7.5 contained reproducibly homogeneous communities dominated by one amoA RFLP type from the N . europaea-N . eutropha group . Duplicate reactors at pH 7.0 developed into diverse communities and showed transient population changes even within the ammonia oxidizer community . Reactors at pH 7.5 and 25°C formed communities that were indistinguishable by the applied FISH probes but differing in amoA RFLP types . Communities in reactors fed with sludge digester supernatant exhibited a higher diversity and were constantly reinoculated with ammonium oxidizers from the supernatant . Therefore, such systems could be maintained at a higher dilution rate (0.75 day-1 compared to 0.2 day-1 for the synthetic wastewater reactors) . Despite similar reactor performance with respect to chemical parameters, the underlying community structures were different, which may have an influence on stability during perturbations .
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