Molecular analysis of fusidic acid resistance in Staphylococcus aureus

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.     

Drebrin particles: components in the ensemble of proteins regulating actin dynamics of lamellipodia and filopodia

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.     

The Fugu rubripes tyrosinase gene promoter targets transgene expression to pigment cells in the mouse

The regulation of the mouse tyrosinase gene expression is controlled by a highly conserved element at -100 bp, the M-box, and an enhancer at -12 kb. In most vertebrates, the length of intergenic sequences makes it difficult to analyze the whole gene and the complete regulatory region. We took advantage of the compact Fugu genome to identify regulatory regions involved in pigment cell-specific expression. We isolated the Fugu tyrosinase gene, and identified putative cis-acting regulatory elements within the promoter. We then asked whether the Fugu promoter sequence functions in mouse pigment cells. We showed that E11.5 transgenic embryos bearing 6 kb or 3 kb of Fugu tyrosinase 5' sequence fused to the reporter gene lacZ revealed melanoblast and RPE-specific expression. This is the first evidence that the tyrosinase promoter is active at midgestation in melanoblasts, long before the onset of pigmentation.     

Transgenic reexpression of GLUT1 or GLUT2 in pancreatic beta cells rescues GLUT2-null mice from early death and restores normal glucose-stimulated insulin secretion

GLUT2-null mice are hyperglycemic, hypoinsulinemic, hyperglucagonemic, and glycosuric and die within the first 3 weeks of life. Their endocrine pancreas shows a loss of first phase glucose-stimulated insulin secretion (GSIS) and inverse alpha to beta cell ratio. Here we show that reexpression by transgenesis of either GLUT1 or GLUT2 in the pancreatic beta cells of these mice allowed mouse survival and breeding. The rescued mice had normal-fed glycemia but fasted hypoglycemia, glycosuria, and an elevated glucagon to insulin ratio. Glucose tolerance was, however, normal. In vivo, insulin secretion assessed following hyperglycemic clamps was normal. In vitro, islet perifusion studies revealed that first phase of insulin secretion was restored as well by GLUT1 or GLUT2, and this was accompanied by normalization of the glucose utilization rate. The ratio of pancreatic insulin to glucagon and volume densities of alpha to beta cells were, however, not corrected. These data demonstrate that 1) reexpression of GLUT1 or GLUT2 in beta cells is sufficient to rescue GLUT2-null mice from lethality, 2) GLUT1 as well as GLUT2 can restore normal GSIS, 3) restoration of GSIS does not correct the abnormal composition of the endocrine pancreas. Thus, normal GSIS does not depend on transporter affinity but on the rate of uptake at stimulatory glucose concentrations.     

Force, speed, and oxygen consumption in Thoroughbred and draft horses

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.

     

Minimal sample requirement for highly multiplexed protein quantification in cell lines and tissues by PCT‐SWATH mass spectrometry

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.