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Biomedical subjects

B L Shapiro

Publications and source records attributed to B L Shapiro.

At least 37 records · Page 2Linked to original sources
PubMed1982

Calcium and age in fibroblasts from control subjects and patients with cystic fibrosis.

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.

Adolescent↗
PubMed1982

The calcium abnormality in cystic fibrosis mitochondria: relative role of respiration and ATP hydrolysis.

Calcium uptake by mitochondria isolated from skin fibroblasts of patients with cystic fibrosis and controls was studied in the presence and absence of inhibitors. Since mitochondrial calcium accumulation may be supported by ATP hydrolysis or respiration, inhibitors of each were used to characterize the basis of previously described alterations in calcium uptake by mitochondria from patients with cystic fibrosis. Calcium uptake measurements under the influence of oligomycin and antimycin A suggest that the increased calcium uptake by mitochondria from patients with cystic fibrosis is related to altered respiratory system activity. Binding constants of calcium to the carrier system in mitochondria were not different between genotypes.

Adenosine Triphosphate↗
PubMed1982

Mitochondrial NADH dehydrogenase in cystic fibrosis: enzyme kinetics in cultured fibroblasts.

Differences among cystic fibrosis (CF) genotypes (CF, obligate carriers for CF [HZ], and controls) in mitochondrial calcium pool size, oxygen (O2) consumption, and rotenone inhibition of O2 consumption led to examination of mitochondrial NADH dehydrogenase (NADH: [acceptor] oxidoreductase, E.C. 1.6.99.3). pH optima of mitochondrial NADH dehydrogenase were different in enzyme derived from whole cell homogenates of cultured skin fibroblasts of subjects with CF, HZ, and controls. We describe here apparent binding of substrate to the enzyme (Km [NADH]) in cell fractions. Km (NADH) for CF ranged from 10.9 to 16.1 micro M (no. = 7); for HZ from 20.9 to 26.3 microM (no. = 5). With three exceptions, Km for controls (no. = 12) ranged from 31.8 to 42.8 microM. Km of the three exceptional controls were 21.5, 23.7, and 22.4 microM (the latter two are identical twins). pH optima of enzyme from these three strains were no different from that of known HZ. The correlation between two kinetic parameters of an enzyme and the three CF genotypes suggests an association between the CF gene and mitochondrial NADH dehydrogenase.

Adolescent↗
PubMed1981

One to one correlation of histological and histochemical light microscopy with scanning electron microscopy.

Several histological stains were applied to specimens after scanning electron microscopic (SEM) processing. Histochemical stains were applied before SEM fixation. After staining, the specimens were processed and dried by SEM techniques. The specimens were taped to a microslide, specimen side up, scribed and covered with immersion oil. After light micrography (LM), the oil was removed and the specimens mounted and gold coated. The same cells were then relocated and photographed by SEM. Periodic acid Schiff was not usable as a specific tissue aldehyde stain, but did prove to be a useful counterstain for dehydrogenase stained specimens. Colloidal iron, as seen by SEM, resulted in a non-specific granular deposit over the specimen and the substrate. All the other histological stains examined--alcian blue, Grams, Feulgen and toluidine blue--were specific and did not change the specimen ultrastructure. The histochemical stains--acid and alkaline phosphatase and three dehydrogenases--were also specific. There were some minor ultrastructural changes with the AcPase and AlPase stains. However, the tissue surface was not significantly distorted. These and other techniques should prove to be a useful adjunct to SEM studies.

Amniotic Fluid↗
PubMed1980

The effect of alloxan diabetes and insulin on the rate of protein synthesis in the rat submandibular gland.

The incorporation of radioactively labeled leucine into TCA-precipitable proteins by submandibular gland tissue slices from control, alloxan diabetic, and insulin supplemented diabetic rats was measured in vitro. Incorporation decreased in alloxan diabetes and could be restored to control levels within three hours after insulin administration. The effects of alloxan diabetes and insulin on 3H-leucine incorporation paralleled their effects on a secretory enzyme, peroxidase. Insulin in vitro stimulated the incorporation of 3H-leucine within 15 minutes of addition to the incubation medium. Further, the response to insulin was found to be dose-related. The conclusion drawn from these results is that insulin has a rapid, direct effect on the rate of protein synthesis in the rat submandibular gland.

Animals↗
PubMed1979

Premature senescence in cultured skin fibroblasts from subjects with cystic fibrosis.

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.

Adolescent↗
PubMed1979

Mitrochondrial NADH dehydrogenase in cystic fibrosis.

We have shown that skin fibroblast from patients with cystic fibrosis (CF) and from carriers for CF [heterozygotes (HZ)] consume more O2 than do their controls. When the mitochondrial electron transport inhibitor rotenone was added to the cells, the relative inhibition of O2 consumption was CF greater than HZ greater than controls (P less than 0.005 in both comparisons). Because rotenone specifically inhibits NADH dehydrogenase, [NADH: (acceptor) oxidoreductase, EC 1.6.99.3], 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 greater than HZ = controls; at pH 8.6, affinity was CF greater than HZ = controls (P less than 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.

Cells, Cultured↗
PubMed1979

Scanning electron microscopy of uncoated human metaphase chromosomes.

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.

Aluminum↗
PubMed1979

Altered intracellular calcium in fibroblasts from patients with cystic fibrosis and heterozygotes.

The importance of intracellular calcium (Ca) in secretion and transmembrane ion movement led us to study Ca in cells from patients with cystic fibrosis (CF) which is a lethal genetic exocrinopathy. Skin fibroblasts from patients with CF, obligate heterozygotes (HZ), and age- and sex-matched controls (C) were used in matched pair experiments measuring 45Ca exchange into and efflux from the cells over time. CF cell lines and HZ cell lines exhibit increased 45Ca exchange when compared with their respective controls (P less than 0.005). The magnitude of this difference (approximately 30%) is not reduced when cells are washed with lanthanum chloride after the exchange period. This difference is likely attributable to an altered capacity of one or more of the intracellular Ca sequestering organelles. Further evidence for this explanation was seen in 45Ca efflux experiments in which CF cells retained a higher percent of their initial 0-time 45Ca than did C cells late in the efflux period (P less than 0.05). The finding of an altered Ca pool size in both CF and particularly HZ cells suggests that altered Ca metabolism is related to the basic gene defect in CF.

Body Fluids↗
PubMed1978

Doubling time alpha-aminoisobutyrate transport and calcium exchange in cultured fibroblasts from cystic fibrosis and control subjects.

Population doubling time, kinetics of transport of alpha-aminoisobutyrate (AIB) and calcium (Ca) exchange were studied in skin fibroblast monolayers obtained from 5 subjects with cystic fibrosis (CF) and 5 age- and sex-matched controls. Population doubling time as estimated from cell count, protein and DNA was no different in the two groups. KM, Vmax, maximal uptake and time of half maximal uptake of AIB were no different in the two groups. Intracellular Ca pool size based on exchange of 45Ca with unlabelled Ca was significantly greater in monolayers from CF subjects.

Aminoisobutyric Acids↗
PubMed1977

Rapid preparation of uncoated biological specimens for scanning electron microscopy.

We have developed a relatively rapid glutaraldehyde-tannic acid (GTA) and osmium tetroxide (OsO4) fixation procedure which permits many types of uncoated biological specimens to be examined in the scanning electron microscope (SEM) at 20 kV without the occurrence of charging. Most specimens taken one day can be examined in the SEM the following afternoon. Types of specimens successfully treated were perfused adult and embryonic rat tissues, confluent human skin fibroblast tissue cultures, plant roots, flowers, seeds, some garden insects, and microcolonies of salivary streptococci. Cells in suspension and extracted human teeth did become electron conductive when treated with the GTA procedure. Most suspended cells must be centrifuged between each solution and the GTA procedure increases the preparation time for these cells. Extracted teeth are usually simply dried and coated. Therefore, the usual SEM preparation techniques are shorter and perhaps more useful for these types of specimens.

Animals↗
PubMed1976

Cellular glycosaminoglycans in lymphocytes from patients with cystic fibrosis.

Cellular glycosaminoglycans were isolated from lymphocytes from patients with cystic fibrosis and controls. The isolated glycosaminoglycans were fractionated by cellulose acetate electrophoresis, analyzed for glucosamine and galactosamine content, and subjected to hydrolysis with bovine testicular hyaluronidase. The total glycosaminoglycan content, the per cent glucosamine and galactosamine, and the distribution of cellular glycosaminoglycans in circulating lymphocytes in cystic fibrosis were no different from controls.

Cystic Fibrosis↗