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Intracellular and Plasma Pharmacokinetics of Saquinavir-Ritonavir, Administered at 1,600/100 Milligrams Once Daily in Human Immunodeficiency Virus-Infected Patients. Jennifer Ford, 2004.Ritonavir-boosted saquinavir (SQV/r) is currently licensed as a twice-daily regimen . Reducing the pill burden with once-daily dosing may improve adherence . Intracellular concentrations of drugs must be related to the clinical efficacy of protease inhibitors . The aims of the study were to determine the cellular and plasma saquinavir and ritonavir concentrations, to determine the half-lives (t1/2s) of the drugs in each compartment, and to examine relationships between drug accumulation and lymphocyte subset P glycoprotein (P-gp) expression . Venous blood samples from 12 human immunodeficiency virus-infected patients receiving a hard-gel formulation of SQV/r (1,600/100 mg once daily) were collected at 2, 6, 12, and 24 h after dosing . Peripheral blood mononuclear cells were separated by density gradient centrifugation, and P-gp expression was measured by dual-color flow cytometry . Plasma and intracellular (cell-associated) drug concentrations were measured by high-performance liquid chromatography-tandem mass spectrometry . The ratio of the intracellular drug area under the concentration-time curve from 0 to 24 h (AUC0-24 h) to plasma drug AUC0-24 h was calculated to determine cellular drug accumulation . The median (range) AUC0-24 h of saquinavir in plasma was 16.2 (5.7 to 39.3) mg · h · liter–1, and that in cells was 46.3 (24.7 to 114.6) mg · h · liter–1 . Corresponding ritonavir values were 7.5 (1.5 to 14.6) mg · h · liter–1 and 10.4 (3.2 to 13.7) mg · h · liter–1, respectively . The median accumulation ratios of cellular AUC to plasma AUC for saquinavir and ritonavir were 3.31 (range, 1.49 to 6.69) and 1.46 (range, 0.83 to 4.15), respectively . Significant differences between the plasma and intracellular saquinavir t1/2s (4.5 h [range, 2.5 to 9.3 h] and 5.9 h [range, 4.0 to 17.7 h]; P = 0.034) and between the plasma and intracellular ritonavir t1/2s (4.1 h [range, 2.6 to 8.3 h] and 6.2 h [range, 3.9 to 18.6 h]; P = 0.032) were observed . No relationship was observed between the accumulation of saquinavir or ritonavir and lymphocyte subset P-gp expression . The intracellular t1/2s of saquinavir and ritonavir were longer than the plasma t1/2s, indicating that intracellular drug may be available at a time when concentrations in plasma are below the minimum effective concentration . Functional Characterization and Regulation of gadX, a Gene Encoding an AraC/XylS-Like Transcriptional Activator of the Escherichia coli Glutamic Acid Decarboxylase System. Angela Tramonti, 2002.The Escherichia coli chromosome contains two distantly located genes, gadA and gadB, which encode biochemically undistinguishable isoforms of glutamic acid decarboxylase (Gad) . The Gad reaction contributes to pH homeostasis by consuming intracellular H+ and producing  -aminobutyric acid . This compound is exported via the protein product of the gadC gene, which is cotranscribed with gadB . Here we demonstrate that transcription of both gadA and gadBC is positively controlled by gadX, a gene downstream of gadA, encoding a transcriptional regulator belonging to the AraC/XylS family . The gadX promoter encompasses the 67-bp region preceding the gadX transcription start site and contains both RpoD and RpoS putative recognition sites . Transcription of gadX occurs in neutral rich medium upon entry into the stationary phase and is increased at acidic pH, paralleling the expression profile of the gad structural genes . However, PT5lacO-controlled gadX expression in neutral rich medium results in upregulation of target genes even in exponential phase, i.e., when the gad system is normally repressed . Autoregulation of the whole gad system is inferred by the positive effect of GadX on the gadA promoter and gadAX cotranscription . Transcription of gadX is derepressed in an hns mutant and strongly reduced in both rpoS and hns rpoS mutants, consistent with the expression profile of gad structural genes in these genetic backgrounds . Gel shift and DNase I footprinting analyses with a MalE-GadX fusion protein demonstrate that GadX binds gadA and gadBC promoters at different sites and with different binding affinities .
Enhanced Expression of S-Adenosylmethionine Synthetase Causes Overproduction of Actinorhodin in Streptomyces coelicolor A3(2). Susumu Okamoto, 2003.We found that a 46-kDa protein is highly expressed in an actinorhodin-overproducing Streptomyces coelicolor A3(2) mutant (KO-179), which exhibited a low-level resistance to streptomycin . The protein was identified as S-adenosylmethionine (SAM) synthetase, which is a product of the metK gene . Enzyme assay revealed that SAM synthetase activity in strain KO-179 was 5- to 10-fold higher than in wild-type cells . The elevation of SAM synthetase activity was found to be associated with an increase in the level of intracellular SAM . RNase protection assay revealed that the metK gene was transcribed from two distinct promoters (p1 and p2) and that enhanced expression of the MetK protein in the mutant strain KO-179 was attributed to elevated transcription from metKp2 . Strikingly, the introduction of a high-copy-number plasmid containing the metK gene into wild-type cells resulted in a precocious hyperproduction of actinorhodin . Furthermore, the addition of SAM to the culture medium induced Act biosynthesis in wild-type cells . Overexpression of metK stimulated the expression of the pathway-specific regulatory gene actII-ORF4, as demonstrated by the RNase protection assay . The addition of SAM also caused hyperproduction of streptomycin in Streptomyces griseus . These findings implicate the significant involvement of intracellular SAM in initiating the onset of secondary metabolism in Streptomyces . Methionine Regeneration and Aminotransferases in Bacillus subtilis, Bacillus cereus, and Bacillus anthracis. Bradley J. Berger, 2003.The conversion of ketomethiobutyrate to methionine has been previously examined in a number of organisms, wherein the aminotransferases responsible for the reaction have been found to be members of the Ia subfamily (L . C . Berger, J . Wilson, P . Wood, and B . J . Berger, J . Bacteriol . 183:4421-4434, 2001) . The genome of Bacillus subtilis has been found to contain no subfamily Ia aminotransferase sequences . Instead, the analogous enzymes in B . subtilis were found to be members of the If subfamily . These putative aspartate aminotransferases, the yugH, ywfG, ykrV, aspB, and patA gene products, have been cloned, expressed, and characterized for methionine regeneration activity . Only YkrV was able to convert ketomethiobutyrate to methionine, and it catalyzed the reaction only when glutamine was used as amino donor . In contrast, subcellular homogenates of B . subtilis and Bacillus cereus utilized leucine, isoleucine, valine, alanine, phenylalanine, and tyrosine as effective amino donors . The two putative branched-chain aminotransferase genes in B . subtilis, ybgE and ywaA, were also cloned, expressed, and characterized . Both gene products effectively transaminated branched-chain amino acids and ketoglutarate, but only YbgE converted ketomethiobutyrate to methionine . The amino donor preference for methionine regeneration by YbgE was found to be leucine, isoleucine, valine, phenylalanine, and tyrosine . The B . subtilis ybgE gene is a member of the family III of aminotransferases and falls in a subfamily designated here IIIa . Examination of B . cereus and Bacillus anthracis genome data found that there were no subfamily IIIa homologues in these organisms . In both B . cereus and B . anthracis, two putative branched-chain aminotransferases and two putative D-amino acid aminotransferases were discovered as members of subfamily IIIb . These four sequences were cloned from B . cereus, expressed, and characterized . Only the gene product from the sequence designated Bc-BCAT2 was found to convert ketomethiobutyrate to methionine, with an amino donor preference of leucine, isoleucine, valine, phenylalanine, and tyrosine . The B . anthracis homologue of Bc-BCAT2 was also cloned, expressed, and characterized and was found to be identical in activity . The aminooxy compound canaline was found to be an uncompetitive inhibitor of B . subtilis YbgE and also inhibited growth of B . subtilis and B . cereus in culture . Nondigestible Oligosaccharides Enhance Bacterial Colonization Resistance against Clostridium difficile In Vitro. Mark J. Hopkins, 2003.Clostridium difficile is the principal etiologic agent of pseudomembranous colitis and is a major cause of nosocomial antibiotic-associated diarrhea . A limited degree of success in controlling C . difficile infection has been achieved by using probiotics; however, prebiotics can also be used to change bacterial community structure and metabolism in the large gut, although the effects of these carbohydrates on suppression of clostridial pathogens have not been well characterized . The aims of this study were to investigate the bifidogenicity of three nondigestible oligosaccharide (NDO) preparations in normal and antibiotic-treated fecal microbiotas in vitro and their abilities to increase barrier resistance against colonization by C . difficile by using cultural and molecular techniques . Fecal cultures from three healthy volunteers were challenged with a toxigenic strain of C . difficile, and molecular probes were used to monitor growth of the pathogen, together with growth of bifidobacterial and bacteroides populations, over a time course . Evidence of colonization resistance was assessed by determining viable bacterial counts, short-chain fatty acid formation, and cytotoxic activity . Chemostat studies were then performed to determine whether there was a direct correlation between bifidobacteria and C . difficile suppression . NDO were shown to stimulate bifidobacterial growth, and there were concomitant reductions in C . difficile populations . However, in the presence of clindamycin, activity against bifidobacteria was augmented in the presence of NDO, resulting in a further loss of colonization resistance . In the absence of clindamycin, NDO enhanced colonization resistance against C . difficile, although this could not be attributed to bifidobacterium-induced inhibitory phenomena .
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