We have shown that skin fibroblasts from patients with cystic fibrosis (CF) and from carriers for CF [heterozygotes (HZ)] consume more 02 than do their controls. When the mitochondrial electron transport inhibitor rotenone was added to the cells, the relative inhibition of 02 consumption was CF > HZ > controls (P < 0.005 in both comparisons). Because rotenone specifically inhibits NADH dehydrogenase, [NADH:(acceptor) oxidoreductase, EC 1.6.99.31, which is the enzyme of energy-conserving site 1 of the mitochondrial electron transport system, activity and kinetics of this enzyme system were studied in fibroblast homogenates. NADH dehydrogenase activity was equal in cells from the three genotypes. At pH 8.0, affinity of the enzyme for its substrate was CF < HZ = controls; at pH 8.6, affinity was CF> HZ = controls (P < 0.005 for the differences). pH optima for the genotypes were without exception 8.6 (CF), 8.3 (HZ), and 8.0 (control). HZ and control lines were distinguished unequivocally in a blind test on the basis of differences in pH optima. Purified mitochondrial preparations revealed pH optima identical to those found in whole cell homogenates. These data suggest that the mutant gene responsible for CF is expressed in the complex mitochondrial NADH dehydrogenase system.

Cultured skin fibroblasts from subjects with cystic fibrosis exhibited normal population doubling times in early passages. After about 13 cumulative population doublings, cystic fibrosis lines doubled more slowly than controls and ceased doubling after about 19 weekly passages. Control lines continued doubling for 27 passages. The premature senescence noted in cells from subjects with cystic fibrosis reconciles controversial observations of cell doubling reported in the literature. Data presented here demonstrate that experiments with cystic fibrosis cells in late passage may generate misleading results since differences from control lines may be ascribed to generalized senile changes rather than to specific results of the cystic fibrosis genotype.

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Intracellular calcium increases significantly as human fibroblasts age in culture. The calcium increase occurs 5 to 6 weeks (passages) earlier and is significantly greater in fibroblasts from subjects with cystic fibrosis in comparison with cells from control subjects. Intracellular calcium, which is thought to be a pathogenetic factor in cystic fibrosis, may also be a meaningful marker in cell aging.

Human metaphase chromosomes were processed with a 3% glutaraldehydetannic acid technique and examined in a scanning electron microscope at 20 kV either without added metal coating or with 2 nm of sputtered gold coating. Several substrates--aluminum mnium foil, silver mirror deposit and sputtered gold-provided good conductive backgrounds for chromosomal spreads. Silver mirror deposit was the best conductive substrate tested. This method should prove to be a useful tool for monitoring the three-dimensional morphology of mitotic chromosomes with the possibility of studying various banding techniques, chromosomal uncoiling and secondary constrictions currently being examined in chromosomal studies.