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Biological Effects of Short-Term or Prolonged Administration of 9-[2-(Phosphonomethoxy)Propyl]Adenine (Tenofovir) to Newborn and Infant Rhesus Macaques. Koen K. A. Van Rompay, 2004.The reverse transcriptase inhibitor 9-[2-(phosphonomethoxy)propyl]adenine (PMPA; tenofovir) was previously found to offer strong prophylactic and therapeutic benefits in an infant macaque model of pediatric human immunodeficiency virus (HIV) infection . We now summarize the toxicity and safety of PMPA in these studies . When a range of PMPA doses (4 to 30 mg/kg of body weight administered subcutaneously once daily) was administered to 39 infant macaques for a short period of time (range, 1 day to 12 weeks), no adverse effects on their health or growth were observed; this included a subset of 12 animals which were monitored for more than 2 years . In contrast, daily administration of a high dose of PMPA (30 mg/kg subcutaneously) for prolonged periods of time (>8 to 21 months) to 13 animals resulted in a Fanconi-like syndrome (proximal renal tubular disorder) with glucosuria, aminoaciduria, hypophosphatemia, growth restriction, bone pathology (osteomalacia), and reduced clearance of PMPA . The adverse effects were reversible or were alleviated following either complete withdrawal of PMPA treatment or reduction of the daily regimen from 30 mg/kg to 2.5 to 10 mg/kg subcutaneously . Finally, to evaluate the safety of a prolonged low-dose treatment regimen, two newborn macaques were started on a 10-mg/kg/day subcutaneous regimen; these animals are healthy and have normal bone density and growth after 5 years of daily treatment . In conclusion, our findings suggest that chronic daily administration of a high dose of PMPA results in adverse effects on kidney and bone, while short-term administration of relatively high doses and prolonged low-dose administration are safe . Composition and Diversity of Microbial Communities Recovered from Surrogate Minerals Incubated in an Acidic Uranium-Contaminated Aquifer. Catherine L. Reardon, 2004.Our understanding of subsurface microbiology is hindered by the inaccessibility of this environment, particularly when the hydrogeologic medium is contaminated with toxic substances . In this study, surrogate geological media contained in a porous receptacle were incubated in a well within the saturated zone of a pristine region of an aquifer to capture populations from the extant communities . After an 8-week incubation, the media were recovered, and the microbial community that developed on each medium was compared to the community recovered from groundwater and native sediments from the same region of the aquifer, using 16S DNA coding for rRNA (rDNA)-based terminal restriction fragment length polymorphism (T-RFLP) . The groundwater and sediment communities were highly distinct from one another, and the communities that developed on the various media were more similar to groundwater communities than to sediment communities . 16S rDNA clone libraries of communities that developed on particles of a specular hematite medium incubated in the same well as the media used for T-RFLP analysis were compared with those obtained from an acidic, uranium-contaminated region of the same aquifer . The hematite-associated community formed in the pristine area was highly diverse at the species level, with 25 distinct phylotypes identified, the majority of which (73%) were affiliated with the ß-Proteobacteria . Similarly, the hematite-associated community formed in the contaminated area was populated in large part by ß-Proteobacteria (62%); however, only 13 distinct phylotypes were apparent . The three numerically dominant clones from the hematite-associated community from the contaminated site were affiliated with metal- and radionuclide-tolerant or acidophilic taxa, consistent with the environmental conditions . Only two populations were common to both sites . From Genetic Footprinting to Antimicrobial Drug Targets: Examples in Cofactor Biosynthetic Pathways. Svetlana Y. Gerdes, 2002.Novel drug targets are required in order to design new defenses against antibiotic-resistant pathogens . Comparative genomics provides new opportunities for finding optimal targets among previously unexplored cellular functions, based on an understanding of related biological processes in bacterial pathogens and their hosts . We describe an integrated approach to identification and prioritization of broad-spectrum drug targets . Our strategy is based on genetic footprinting in Escherichia coli followed by metabolic context analysis of essential gene orthologs in various species . Genes required for viability of E . coli in rich medium were identified on a whole-genome scale using the genetic footprinting technique . Potential target pathways were deduced from these data and compared with a panel of representative bacterial pathogens by using metabolic reconstructions from genomic data . Conserved and indispensable functions revealed by this analysis potentially represent broad-spectrum antibacterial targets . Further target prioritization involves comparison of the corresponding pathways and individual functions between pathogens and the human host . The most promising targets are validated by direct knockouts in model pathogens . The efficacy of this approach is illustrated using examples from metabolism of adenylate cofactors NAD(P), coenzyme A, and flavin adenine dinucleotide . Several drug targets within these pathways, including three distantly related adenylyltransferases (orthologs of the E . coli genes nadD, coaD, and ribF), are discussed in detail . Microviridae, a Family Divided: Isolation, Characterization, and Genome Sequence of  MH2K, a Bacteriophage of the Obligate Intracellular Parasitic Bacterium Bdellovibrio bacteriovorus.Karie L. Brentlinger, 2002.A novel single-stranded DNA phage, MH2K, of Bdellovibrio bacteriovorus was isolated, characterized, and sequenced . This phage is a member of the Microviridae, a family typified by bacteriophage
X174 . Although B . bacteriovorus and Escherichia coli are both classified as proteobacteria,
MH2K is only distantly related to
X174 . Instead,
MH2K exhibits an extremely close relationship to the Microviridae of Chlamydia in both genome organization and encoded proteins . Unlike the double-stranded DNA bacteriophages, for which a wide spectrum of diversity has been observed, the single-stranded icosahedral bacteriophages appear to fall into two distinct subfamilies . These observations suggest that the mechanisms driving single-stranded DNA bacteriophage evolution are inherently different from those driving the evolution of the double-stranded bacteriophages .
Temperature-Dependent Hypermutational Phenotype in recA Mutants of Thermus thermophilus HB27. Pablo Castán, 2003.The recA gene from Thermus thermophilus HB27 was cloned and engineered to obtain insertion (recA::kat) and deletion (  recA) derivatives . Transcription of recA in this extreme thermophile was induced by mitomycin C, leading to the synthesis of a monocistronic mRNA . This DNA damage-mediated induction was dependent on the integrity of recA. In addition to UV sensitivity, the recA mutants of T . thermophilus showed severe pleiotropic defects, ranging from irregular nucleoid condensation and segregation to a dramatic reduction in viability during culture . An increase in the frequency of both carotenoidless and auxotrophic mutants within surviving cells of the
recA strain indicated a high mutation rate . As RecA is not required for plasmid transformation, we have used the
 -lacZ gene fragment and the ampicillin resistance gene from Escherichia coli as passenger reporters to confirm such high mutation rates . Our data support the idea that the absence of RecA results in a hypermutational phenotype in T . thermophilus . Furthermore, a direct relationship is deduced between the growth temperature and mutation rate, which finally has a deleterious effect on cell survival in the absence of RecA .
Analysis of DNA Binding and Transcriptional Activation by the LysR-Type Transcriptional Regulator CbbR of Xanthobacter flavus. Geertje van Keulen, 2003.The LysR-type transcriptional regulator CbbR controls the expression of the cbb and gap-pgk operons in Xanthobacter flavus, which encode the majority of the enzymes of the Calvin cycle required for autotrophic CO2 fixation . The cbb operon promoter of this chemoautotrophic bacterium contains three potential CbbR binding sites, two of which partially overlap . Site-directed mutagenesis and subsequent analysis of DNA binding by CbbR and cbb promoter activity were used to show that the potential CbbR binding sequences are functional . Inverted repeat IR1 is a high-affinity CbbR binding site . The main function of this repeat is to recruit CbbR to the cbb operon promoter . In addition, it is required for negative autoregulation of cbbR expression . IR3 represents the main low-affinity binding site of CbbR . Binding to IR3 occurs in a cooperative manner, since mutations preventing the binding of CbbR to IR1 also prevent binding to the low-affinity site . Although mutations in IR3 have a negative effect on the binding of CbbR to this site, they result in an increased promoter activity . This is most likely due to steric hindrance of RNA polymerase by CbbR since IR3 partially overlaps with the -35 region of the cbb operon promoter . Mutations in IR2 do not affect the DNA binding of CbbR in vitro but have a severe negative effect on the activity of the cbb operon promoter . This IR2 binding site is therefore critical for transcriptional activation by CbbR . Engineering of Carbon Distribution between Glycolysis and Sugar Nucleotide Biosynthesis in Lactococcus lactis. Ingeborg C. Boels, 2003.We describe the effects of modulating the activities of glucokinase, phosphofructokinase, and phosphoglucomutase on the branching point between sugar degradation and the biosynthesis of sugar nucleotides involved in the production of exopolysaccharide biosynthesis by Lactococcus lactis . This was realized by using a described isogenic L . lactis mutant with reduced enzyme activities or by controlled expression of the well-characterized genes for phosphoglucomutase or glucokinase from Escherichia coli or Bacillus subtilis, respectively . The role of decreased metabolic flux was studied in L . lactis strains with decreased phosphofructokinase activities . The concomitant reduction of the activities of phosphofructokinase and other enzymes encoded by the las operon (lactate dehydrogenase and pyruvate kinase) resulted in significant changes in the concentrations of sugar-phosphates . In contrast, a >25-fold overproduction of glucokinase resulted in 7-fold-increased fructose-6-phosphate levels and 2-fold-reduced glucose-1-phosphate and glucose-6-phosphate levels . However, these increased sugar-phosphate concentrations did not affect the levels of sugar nucleotides . Finally, an  100-fold overproduction of phosphoglucomutase resulted in 5-fold-increased levels of both UDP-glucose and UDP-galactose . While the increased concentrations of sugar-phosphates or sugar nucleotides did not significantly affect the production of exopolysaccharides, they demonstrate the metabolic flexibility of L . lactis.
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