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Caspofungin Uptake Is Mediated by a High-Affinity Transporter in Candida albicans. Padmaja Paderu, 2004.The uptake of the echinocandin drug caspofungin acetate in Candida albicans was evaluated at drug levels at or near the MIC for the organism . Maximal uptake was achieved in 10 min and was energy independent . A saturable transport system, consistent with a facilitated-diffusion carrier, was observed with the unlabeled drug competing with the labeled drug for uptake and efflux . More than 90% of the transported drug was observed in a single kinetic compartment that was available for efflux, indicating that the drug was free in the cytoplasm following uptake . Efflux was also energy independent but was sensitive to the presence of a fully loaded carrier on both faces of the bilayer . Overall, the data presented are consistent with the presence of a high-affinity facilitated-diffusion transporter that mediates caspofungin uptake and could be a potential source of transport-related reduced susceptibility . Physiological Characterization of a Heme-Deficient Mutant of Staphylococcus aureus by a Proteomic Approach. Christian Kohler, 2003.The high-resolution two-dimensional (2D) protein gel electrophoresis technique combined with matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for identification of proteins whose levels were changed by a mutation in hemB . Cytoplasmic protein extracts obtained from the mutant and the wild type (strain COL) at different stages of growth in tryptone soya broth (exponential, transitional, and stationary growth phases) were separated on 2D protein gels . Comparison of the 2D patterns of the protein extracts of the two strains revealed major differences . Because the electron transport chain of the mutant is interrupted due to the deficiency of heme, this organism should be unable to use oxygen or nitrate as a terminal electron acceptor . Consistent with this hypothesis, proteins involved in the glycolytic pathway and related pathways (glyceraldehyde-3-phosphate dehydrogenase, enolase, and phosphoglycerate kinase) and in fermentation pathways (lactate dehydrogenase, alcohol dehydrogenase, and pyruvate formate lyase) were induced in exponentially growing cells of the mutant . These results strongly indicate that the hemB mutant generates ATP from glucose or fructose only by substrate phosphorylation . Analyses of the fermentation reactions showed that the main product was lactate . Although pyruvate formate lyase (Pfl) and pyruvate dehydrogenase were present, neither ethanol nor acetate was detected in significant amounts . Presumably, Pfl was not activated in the presence of oxygen, and pyruvate dehydrogenase might have very low activity . Transcriptional analysis of citB, encoding the aconitase, revealed that the activity of the citrate cycle enzymes was down-regulated in the hemB mutant . The arginine deiminase pathway was also induced, and it could provide ATP as well . Furthermore, the amounts of most of the extracellular virulence factors were significantly reduced by a mutation in hemB, which is consistent with previous reports . Engineering Escherichia coli for Increased Productivity of Serine-Rich Proteins Based on Proteome Profiling. Mee-Jung Han, 2003.Variations in proteome profiles of Escherichia coli in response to the overproduction of human leptin, a serine-rich (11.6% of total amino acids) protein, were examined by two-dimensional gel electrophoresis . The levels of heat shock proteins increased, while those of protein elongation factors, 30S ribosomal protein, and some enzymes involved in amino acid biosynthesis decreased, after leptin overproduction . Most notably, the levels of enzymes involved in the biosynthesis of serine family amino acids significantly decreased . Based on this information, we designed a strategy to enhance the leptin productivity by manipulating the cysK gene, encoding cysteine synthase A . By coexpression of the cysK gene, we were able to increase the cell growth rate by approximately twofold . Also, the specific leptin productivity could be increased by fourfold . In addition, we found that cysK coexpression can improve the production of another serine-rich protein, interleukin-12 ß chain, suggesting that this strategy may be useful for the production of other serine-rich proteins as well . The approach taken in this study should be useful in designing a strategy for improving recombinant protein production .
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