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Cellular Pharmacokinetics and Pharmacodynamics of the Glycopeptide Antibiotic Oritavancin (LY333328) in a Model of J774 Mouse Macrophages. Françoise Van Bambeke, 2004.The intracellular pharmacokinetics and pharmacodynamics of oritavancin (LY333328) were studied in cultured cells . Oritavancin was avidly accumulated by J774 and THP-1 macrophages and rat fibroblasts and to a lesser extent by LLC-PK1 and Caco-2 cells . In J774 macrophages, the level of accumulation reached a plateau (at 370-fold the extracellular concentration) within 24 h and was partly defeated by a rise in serum protein levels . Efflux was incomplete (with a plateau at two-thirds of the original level at 6 h) . In short-term kinetic studies, oritavancin uptake was linear for up to 4 h (as was the case for horseradish peroxidase and small latex beads, used as markers of the fluid phase and adsorptive endocytosis, respectively), which was in contrast to azithromycin and chloroquine uptake (which accumulate in cells by diffusion and segregation) . The rates of clearance of oritavancin and latex beads were comparable (150 and 120 µl x mg of protein–1 x h–1, respectively) and were approximately 200 times higher than that of horseradish peroxidase . Oritavancin accumulation was partially reduced by monensin but was unaffected by acidic pH (these conditions abolished chloroquine accumulation) . Cell-associated oritavancin was found in lysosomal fractions after homogenization of J774 macrophages and fractionation by isopycnic centrifugation . Oritavancin was bactericidal against intracellular Staphylococcus aureus (phagolysosomal infection) but was unable to control the intracellular growth of Listeria monocytogenes (cytosolic infection), even though its cellular concentration largely exceeded the MIC (0.02 mg/liter) and minimal bactericidal concentration (2 mg/liter) . We conclude that oritavancin enters cells by adsorptive endocytosis (favored by its lipophilic side chain and/or the presence of three protonatable amines), which drives it to lysosomes, where it exerts antibiotic activity . Identification of Quorum-Sensing-Regulated Genes of Burkholderia cepacia. Claudio Aguilar, 2003.Quorum sensing is a regulatory mechanism (operating in response to cell density) which in gram-negative bacteria usually involves the production of N-acyl homoserine lactones (HSL) . Quorum sensing in Burkholderia cepacia has been associated with the regulation of expression of extracellular proteins and siderophores and also with the regulation of swarming and biofilm formation . In the present study, several quorum-sensing-controlled gene promoters of B . cepacia ATCC 25416 were identified and characterized . A total of 28 putative gene promoters show CepR-C8-HSL-dependent expression, suggesting that quorum sensing in B . cepacia is a global regulatory system . Prokaryotic Metabolic Activity and Community Structure in Antarctic Continental Shelf Sediments. J. P. Bowman, 2003.The prokaryote community activity and structural characteristics within marine sediment sampled across a continental shelf area located off eastern Antarctica (66°S, 143°E; depth range, 709 to 964 m) were studied . Correlations were found between microbial biomass and aminopeptidase and chitinase rates, which were used as proxies for microbial activity . Biomass and activity were maximal within the 0- to 3-cm depth range and declined rapidly with sediment depths below 5 cm . Most-probable-number counting using a dilute carbohydrate-containing medium recovered 1.7 to 3.8% of the sediment total bacterial count, with mostly facultatively anaerobic psychrophiles cultured . The median optimal growth temperature for the sediment isolates was 15°C . Many of the isolates identified belonged to genera characteristic of deep-sea habitats, although most appear to be novel species . Phospholipid fatty acid (PLFA) and isoprenoid glycerol dialkyl glycerol tetraether analyses indicated that the samples contained lipid components typical of marine sediments, with profiles varying little between samples at the same depth; however, significant differences in PLFA profiles were found between depths of 0 to 1 cm and 13 to 15 cm, reflecting the presence of a different microbial community . Denaturing gradient gel electrophoresis (DGGE) analysis of amplified bacterial 16S rRNA genes revealed that between samples and across sediment core depths of 1 to 4 cm, the community structure appeared homogenous; however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident . Sequencing of DGGE bands and rRNA probe hybridization analysis revealed that the major community members belonged to delta proteobacteria, putative sulfide oxidizers of the gamma proteobacteria, Flavobacteria, Planctomycetales, and Archaea . rRNA hybridization analyses also indicated that these groups were present at similar levels in the top layer across the shelf region . Isolation of Strains Belonging to the Cosmopolitan Polynucleobacter necessarius Cluster from Freshwater Habitats Located in Three Climatic Zones. Martin W. Hahn, 2003.More than 40 bacterial strains belonging to the cosmopolitan Polynucleobacter necessarius cluster (Betaproteobacteria) were isolated from a broad spectrum of freshwater habitats located in three climatic zones . Sequences affiliated with the freshwater P . necessarius cluster are among the most frequently detected in studies on bacterial diversity in freshwater ecosystems . Despite this frequent detection with culture-independent techniques and the cosmopolitan occurrence of members affiliated with this cluster, no isolates have been reported thus far . The isolated strains have been obtained from lakes, ponds, and rivers in central Europe, the People's Republic of China, and East Africa by use of the filtration-acclimatization method . The 16S rRNA gene sequences of the isolates are 98.8 to 100% identical to reference sequences obtained by various authors by use of culture-independent methods . The isolates, aerobic heterotrophs, grew on a wide range of standard complex media and formed visible colonies on agar plates . Thus, the previous lack of isolates cannot be explained by a lack of appropriate media . Most of the isolates possess, under a wide range of culture conditions, very small cells (<0.1 µm3), even when grown in medium containing high concentrations of organic substances . Thus, these strains are obligate ultramicrobacteria . The obtained strains have a C-shaped cell morphology which is very similar to that of recently isolated ultramicrobacterial Luna cluster strains (Actinobacteria) and the SAR11 cluster strains (Alphaproteobacteria) .
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