Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein. While 70% of CF chromosomes carry a deletion of the phenylalanine residue 508 (deltaF508) of CFTR, roughly 5% of all CF chromosomes carry a premature stop mutation. We reported that the aminoglycoside antibiotics G-418 and gentamicin can suppress two premature stop mutations [a stop codon in place of glycine residue 542 (G542X) and arginine residue 553 (R553X)] when expressed from a CFTR cDNA in HeLa cells. Suppression resulted in the synthesis of full-length CFTR protein and the appearance of a cAMP-activated anion conductance characteristic of CFTR function. However, it was unclear whether this approach could restore CFTR function in cells expressing mutant forms of CFTR from the nuclear genome. We now report that G-418 and gentamicin are also capable of restoring CFTR expression in a CF bronchial epithelial cell line carrying the CFTR W1282X premature stop mutation (a stop codon in place of tryptophan residue 1282). This conclusion is based on the reappearance of cAMP-activated chloride currents, the restoration of CFTR protein at the apical plasma membrane, and an increase in the abundance of CFTR mRNA levels from the W1282X allele.

Uptake of [14C]choline upon hyperosmotic stress of exponential-phase Staphylococcus aureus cultures in a complex medium occurred after a delay of 2.5 to 3.5 h. This uptake could be prevented by chloramphenicol, suggesting that it occurred via an inducible transport system. Radioactivity from ['4Clcholine was accumulated as [14Clglycine betaine. However, neither choline nor glycine betaine could act as the major carbon and energy source for the organism, suggesting that choline was not metabolized beyond glycine betaine. Assay of choline transport activity in cells grown under different conditions in defined media revealed that osmotic stress was mainly responsible for the induction, but choline gave a further increase in induction. The system was not induced in anaerobically grown cells. Choline transport activity was repressed by glycine betaine and proline betaine, suggesting that these compounds are corepressors. Choline transport activity was not induced in cells osmotically stressed by 1 M potassium phosphate or 0.5 M sodium phosphate, but was induced in cells grown in low-phosphate medium in the absence of osmotic stress. This suggests that there is a connection between the phosphate and osmotic stress regulons. Choline transport was energy and Na' dependent and had a Km of 46 FLM and a maximum rate of transport (Vmax) of 54 nmol/min/mg (dry weight). The results of competition studies suggested that N-methyl and an alcohol group or aldehyde groups at the ends of the molecule were important in its recognition by the system. Glycine betaine was not a highly effective competitor, suggesting that its transport system and the choline transport system were distinct from each other. Choline transport was highly susceptible to a variety of inhibitors, which may be related to the greater dependence on respiratory metabolism of cells grown in the presence of high NaCl concentrations.

Staphylococcus aureus NCTC 8325 exhibited a long lag phase (11 h) when inoculated into defined medium lacking proline, that could be shortened by increasing the concentration of arginine in the medium, or by supplying ornithine. Radioactivity from ~-['~C]arginine, but not ~-[l~C]glutamate was incorporated into a spot with the chromatographic mobility of [14C]proline in the pool metabolites fraction. Selection for transposon TnSI7-lacZ mutants impaired in arginine catabolism yielded four proline auxotrophs. Enzyme assays and precursor feeding experiments suggested that the major pathway for proline biosynthesis in S. aureus was from arginine via ornithine and A'-pyrroline 5-carboxylate, rather than from glutamate. Strain 8325 Pro+, a proline prototrophic variant obtained by cultivation of 8325 in the absence of proline, accumulated ~-[l~C]arginine from the medium at about eight times the rate of strain 8325, suggesting its response to proline starvation was to increase arginine uptake.1

No abstract