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Antifungal Activities of Posaconazole and Granulocyte-Macrophage Colony-Stimulating Factor Ex Vivo and in Mice with Disseminated Infection Due to Scedosporium prolificans. M. Simitsopoulou, 2004.Invasive infection due to Scedosporium prolificans is characterized by drug resistance and a high rate of mortality . The effects of posaconazole (POS), an investigational antifungal triazole, murine granulocyte-macrophage colony-stimulating factor (GM-CSF), and their combination against S . prolificans were evaluated ex vivo and in a newly developed murine model of disseminated infection due to this organism . When POS was combined with polymorphonuclear leukocytes from untreated or GM-CSF-treated mice (P < 0.01) ex vivo, it had increased activity in terms of the percentage of hyphal damage . Immunocompetent BALB/c mice were infected with 4 x 104 conidia of S . prolificans via the lateral tail vein . At 24 h postinfection the mice were treated with GM-CSF (5 µg/kg of body weight/day subcutaneously), POS (50 mg/kg/day by gavage), both agents, or saline only . Half of the brain, lung, liver, and kidney from each animal were cultured; and the other half of each organ was processed for histopathology . The mean survival times were 7.0 ± 0.3 days for the controls, 7.4 ± 0.4 days for POS-treated mice, 8.0 ± 0.3 days for GM-CSF-treated mice (P = 0.08 compared with the results for the controls), and 7.3 ± 0.3 days for POS-GM-CSF-treated mice . Fungal burdens (determined as the numbers of CFU per gram of tissue) were found in descending orders of magnitude in the kidneys, brains, livers, and lungs . The burdens were significantly reduced in the brains of GM-CSF-treated mice (P < 0.05) and the livers of POS-treated mice (P < 0.05) . The numbers of lesions in the organs closely corresponded to the fungal burdens . GM-CSF tended to prolong survival (P = 0.08 compared with the results for the controls) . While the combination of POS and GM-CSF showed enhanced activity ex vivo, it did not increase the activities of the two agents against this highly refractory filamentous fungus in mice . Phage TP901-1 Site-Specific Integrase Functions in Human Cells. Stephanie M. Stoll, 2002.We demonstrate that the site-specific integrase encoded by phage TP901-1 of Lactococcus lactis subsp . cremoris has potential as a tool for engineering mammalian genomes . We constructed vectors that express this integrase in Escherichia coli and in mammalian cells and developed a simple plasmid assay to measure the frequency of intramolecular integration mediated by the integrase . We used the assay to document that the integrase functions efficiently in E . coli and determined that for complete reaction in E . coli, the minimal sizes of attB and attP are 31 and 50 bp, respectively . We carried out partial purification of TP901-1 integrase protein and demonstrated its functional activity in vitro in the absence of added cofactors, characterizing the time course and temperature optimum of the reaction . Finally, we showed that when expressed in human cells, the TP901-1 integrase carries out efficient intramolecular integration on a transfected plasmid substrate in the human cell environment . The TP901-1 phage integrase thus represents a new reagent for manipulating DNA in living mammalian cells . Transcriptional Regulation of the Gene Encoding an Alcohol Dehydrogenase in the Archaeon Sulfolobus solfataricus Involves Multiple Factors and Control Elements. Gabriella Fiorentino, 2003.A transcriptionally active region has been identified in the 5' flanking region of the alcohol dehydrogenase gene of the crenarchaeon Sulfolobus solfataricus through the evaluation of the activity of putative transcriptional regulators and the role of the region upstream of the gene under specific metabolic circumstances . Electrophoretic mobility shift assays with crude extracts revealed protein complexes that most likely contain TATA box-associated factors . When the TATA element was deleted from the region, binding sites for both DNA binding proteins, such as the small chromatin structure-modeling Sso7d and Sso10b (Alba), and transcription factors, such as the repressor Lrs14, were revealed . To understand the molecular mechanisms underlying the substrate-induced expression of the adh gene, the promoter was analyzed for the presence of cis-acting elements recognized by specific transcription factors upon exposure of the cell to benzaldehyde . Progressive dissection of the identified promoter region restricted the analysis to a minimal responsive element (PAL) located immediately upstream of the transcription factor B-responsive element-TATA element, resembling typical bacterial regulatory sequences . A benzaldehyde-activated transcription factor (Bald) that specifically binds to the PAL cis-acting element was also identified . This protein was purified from heparin-fractionated extracts of benzaldehyde-induced cells and was shown to have a molecular mass of  16 kDa . The correlation between S . solfataricus adh gene activation and benzaldehyde-inducible occupation of a specific DNA sequence in its promoter suggests that a molecular signaling mechanism is responsible for the switch of the aromatic aldehyde metabolism as a response to environmental changes .
Dispersal and Phylogenetic Diversity of Nonmarine Picocyanobacteria, Inferred from 16S rRNA Gene and cpcBA-Intergenic Spacer Sequence Analyses. Nicholas D. Crosbie, 2003.More than 20 Synechococcus and Cyanobium isolates were obtained from central European subalpine lakes and sequenced for their 16S rRNA gene and part of the phycocyanin operon (cpc), specifically the intergenic spacer (IGS) between cpcB and cpcA, and corresponding flanking regions (cpcBA-IGS) . Maximum-likelihood analyses revealed the existence of at least six to seven clusters of nonmarine picocyanobacteria within the picophytoplankton clade and support the conjecture of global dispersal for some closely related picocyanobacterial genotypes .
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