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Fusidic acid is a potent antibiotic against severe Gram-positive infections that interferes with the function of elongation factor G (EF-G), thereby leading to the inhibition of bacterial protein synthesis. In this study, we demonstrate that fusidic acid resistance in Staphylococcus aureus results from point mutations within the chromosomal fusA gene encoding EF-G. Sequence analysis of fusA revealed mutational changes that cause amino acid substitutions in 10 fusidic acid-resistant clinical S. aureus strains as well as in 10 fusidic acid-resistant S. aureus mutants isolated under fusidic acid selective pressure in vitro. Fourteen different amino acid exchanges were identified that were restricted to 13 amino acid residues within EF-G. To confirm the importance of observed amino acid exchanges in EF-G for the generation of fusidic acid resistance in S. aureus, three mutant fusA alleles encoding EF-G derivatives with the exchanges P406L, H457Y and L461K were constructed by site-directed mutagenesis. In each case, introduction of the mutant fusA alleles on plasmids into the fusidic acid-susceptible S. aureus strain RN4220 caused a fusidic acid-resistant phenotype. The elevated minimal inhibitory concentrations of fusidic acid determined for the recombinant bacteria were analogous to those observed for the fusidic acid-resistant clinical S. aureus isolates and the in vitro mutants containing the same chromosomal mutations. Thus, the data presented provide evidence for the crucial importance of individual amino acid exchanges within EF-G for the generation of fusidic acid resistance in S. aureus. 相似文献
93.
Wiebke K. Peitsch Ilse Hofmann Silke Prtzel Christine Grund Caecilia Kuhn Ingrid Moll Lutz Langbein Werner W. Franke 《European journal of cell biology》2001,80(9):567-579
Drebrin, an actin-binding 70-kDa protein with an unusually slow SDS-PAGE mobility corresponding to approximately 120 kDa, containing a proline-rich, profilin-binding motif, had originally been reported from neuronal cells, but recently has also been found in diverse other kinds of tissues and cell lines. In biochemical analyses of various cells and tissues, employing gel filtration, sucrose gradient centrifugation, immunoprecipitation and -blotting, we have identified distinct states of soluble drebrin: a approximately 4S monomer, an 8S, ca. 217-kDa putative trimer, a 13S and a > 20S oligomer. In the 8S particles only [35S]methionine-labelled drebrin but no other actin-binding protein has been detected in stoichiometric amounts. By immunofluorescence and immunoelectron microscopy, drebrin-positive material often appeared as "granules" up to 400 nm in diameter, in some cell types clustered near the Golgi apparatus or in lamellipodia, particularly at leading edges, or in dense-packed submembranous masses at tips (acropodia) or ruffles of leading edges, in filopodia and at plaques of adhering junctions. We conclude that these drebrin complexes and drebrin-rich structures allow the build-up and maintenance of high local drebrin concentrations in strategic positions for the regulation of actin filament assembly, thereby contributing to cell motility and morphology, in particular local changes of plasticity and the formation of protrusions. 相似文献
94.
Olschewski H Erlenkötter A Zaborosch C Chemnitius G 《Enzyme and microbial technology》2000,26(7):537-543
Sensors for the determination of L-lysine in samples of fermentation broth have been developed. Low-cost screen-printed sensors comprising a platinum working electrode, an Ag/AgCl pseudo reference and a carbon counter electrode were used as transducers for the enzyme sensors. L-lysine-(alpha)-oxidase from Trichoderma viride has been immobilized by entrapment into a polyurethane hydrogel. Sensors were characterized for L-lysine with respect to pH value, linear range, reproducibility, repeatability, storage and working stability. The sensitivities to other amino acids were also determined. A batch system with two working electrodes, one with immobilized enzyme and one without was adapted for the determination of L-lysine by differential measurements. Good agreement was found between L-lysine concentrations measured by the enzyme sensors and by a conventional amino acid analyzer. 相似文献
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Potard U. Silke Birlenbach; Leith David E.; Fedde M. Roger 《Journal of applied physiology》1998,84(6):2052-2059
Thoroughbred (TB) and draft horses (DH) havelong been selected for tasks of very different intensities andforce-speed relationships. To study their adaptations, we measuredO2 consumption and related variables in three TB and four DH during progressive exercise tests ona level treadmill. The horses exerted a draft force of 0, 5, 10, 15, or20% of their body weight at speeds that increased by 2 m/s every 3 minuntil they could not maintain that speed. We found that TB could exertthe same draft forces as DH and, at each force, TB achieved about twicethe speed, twice the external power, and twice theO2 consumption as DH; thus the twobreeds had the same gross efficiencies. We also found maximalO2 consumption of TB to be abouttwice that of DH (134 vs. 72 ml · kg1 · min1,respectively), suggesting adaptations to high-intensity exercise. Peakefficiency was reached at lower speeds in DH than in TB, suggestingadaptations to high-force, low-speed exercise. These differencesbetween TB and DH in force-speed and aerobic capacities and in speedfor peak efficiency likely reflect different contraction velocities inlocomotor muscles. 相似文献
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Minimal sample requirement for highly multiplexed protein quantification in cell lines and tissues by PCT‐SWATH mass spectrometry
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Shiying Shao Tiannan Guo Chiek Ching Koh Silke Gillessen Markus Joerger Wolfram Jochum Ruedi Aebersold 《Proteomics》2015,15(21):3711-3721
The amount of sample available for clinical and biological proteomic research is often limited and thus significantly restricts clinical and translational research. Recently, we have integrated pressure cycling technology (PCT) assisted sample preparation and SWATH‐MS to perform reproducible proteomic quantification of biopsy‐level tissue samples. Here, we further evaluated the minimal sample requirement of the PCT‐SWATH method using various types of samples, including cultured cells (HeLa, K562, and U251, 500 000 to 50 000 cells) and tissue samples (mouse liver, heart, brain, and human kidney, 3–0.2 mg). The data show that as few as 50 000 human cells and 0.2–0.5 mg of wet mouse and human tissues produced peptide samples sufficient for multiple SWATH‐MS analyses at optimal sample load applied to the system. Generally, the reproducibility of the method increased with decreasing tissue sample amounts. The SWATH maps acquired from peptides derived from samples of varying sizes were essentially identical based on the number, type, and quantity of identified peptides. In conclusion, we determined the minimal sample required for optimal PCT‐SWATH analyses, and found smaller sample size achieved higher quantitative accuracy. 相似文献