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Biomedical subjects

J D Butler

Publications and source records attributed to J D Butler.

49 records · Page 3Linked to original sources
PubMed1980

Cystinotic and normal fibroblasts: differential susceptibility to cysteine toxicity in vitro.

Extracellular cysteine concentrations between 0.5 and 2.5 mM resulted in death of normal but not cystinotic cells grown in Eagle's minimal essential medium containing supplemental fetal bovine serum and antibiotics. Differential cell survival was determined by viable cell counting using Trypan Blue dye exclusion. In cocultivation experiments of [3H]thymidine-labelled cystinotic fibroblasts with nonradioactive normal fibroblasts, autoradiography confirmed the selective survival of cystinotic cells in medium containing 1 mM cysteine. At this concentration of 1 mM cysteine, intracellular cystine content increased slightly in surviving normal cells but not in cystinotic cells, which normally contain a high level of intracellular cystine. This comparative resistance of cystinotic fibroblasts to elevated extracellular cysteine concentrations forms the basis for an in vitro selective system for these mutant human cells. Further exploration of this resistance phenomenon may well expand the understanding of the molecular defect in cystinotic cells.

Cell Count↗
PubMed1979

An appraisal of relative airborne sub-urban concentrations of polycyclic aromatic hydrocarbons monitored indoors and outdoors.

Particle-size distribution studies of polycyclic aromatic hydrocarbons in city and sub-urban atmospheres indicate that these compounds are associated with particles having MMD values of about 0.5 micrometer. Ambient airborne concentrations of pyrene, chrysene, benzo[a]pyrene, benzo[e]pyrene and coronene at suburban sites between 11 and 17 km from a city centre are shown to be approximately similar inside and outside detached residential houses. At all sites, monitored chrysene appears in highest concentration in the range 4-7 ng m-3, followed by benzo[a]pyrene and benzo[e]pyrene between 2-4 ng m-3, with coronene generally less than 1 ng m-3. By taking benzo[a]pyrene as a typical example of this class of compounds, these results indicate that the lungs of sub-urban populations will be exposed to about 34 ng of benzo[a]pyrene per day. This background amount is roughly equivalent to the exposure to this chemical experienced by the lung when one cigarette is smoked per day.

Air Pollutants↗
PubMed1979

Elevated cystine levels in cultured skin fibroblasts from patients with I-cell disease.

Cultured skin fibroblasts from patients with I-cell disease (mucolipidosis II) exhibit multiple deficiency of acid hydrolase activities associated with a defect in the mechanism of packaging of these enzymes into lysosomes. The authors have examined such cells to ascertain whether the impairment of lysosomal function is of so broad a nature as to result in the storage of the amino acid cystine in a manner similar to that seen in cells derived from patients with cystinosis, an unrelated lysosomal storage disease of unknown etiology. Of 10 I-cell lines examined by automated amino acid analysis, seven were found to possess abnormally high levels of total free cyst(e)ine (i.e., greater than 1 nmole 1/2 Cys/mg protein). The mean half-cystine content of those I-cell lines subjected to multiple analysis ranged from 3-10 nmole/mg protein. levels which are comparable to those seen in homozygous cystinotic cells. The cystine content of several of these lines appeared to increase with subculture. Cultured fibroblasts from two patients with the biochemically similar, but clinically less severe, mucolipidosis III (pseudo-Hurler polydystrophy) exhibited normal to marginally elevated levels of cystine, whereas cells from individuals with three different mucopolysaccharide storage disorders contained normal levels of the amino acid. It was concluded that cystine, and not cysteine, was the predominant form of this amino acid in these cells, because previous reaction of I-cell extracts with N-ethylmaleimide did not alter the observed cystine levels. The further identification of excess cystine in these cells was corroborated by analytical results obtained with a highly specific cystine-binding protein method as well as by high-voltage electrophoresis of extracts from cells pulsed with 35S-cystine. Comparative analysis of intracellular amino acids in normal and I-cell fibroblasts indicated that the elevation of csystine seen in the latter was unique to this amino acid and did not reflect a generalized increase in the total free amino acid content of these mutant cells.

Cell Line↗
PubMed1978

Entrapment of metaphase chromosomes into phospholipid vesicles (lipochromosomes): carrier potential in gene transfer.

Transfer of genes from one type of cultured mammalian cell to another by using isolated metaphase chromosomes has been reported with a frequency of one per 10(6)-10(8) cells. Very recently a rate of 16/10(6) has been reported with Chinese hamster ovary cells [Spandidos, D. A. & Siminovitch, L. (1977) Proc. Natl. Acad. Sci. USA 74, 3480-3484]. To increase the frequency of gene transfer, we isolated metaphase chromosomes from hypoxanthine guanine phosphoribosyltransferase (HGPRT) positive cells, entrapped them in liposomes, and fused the lipochromosomes with HGPRT-negative cells. Lipochromosomes were prepared with cholesterol and egg lecithin, using isolated metaphase chromosomes from a mouse-human somatic hybrid cell line (A9/HRBC2); the entire X chromosome, including the HGPRT, glucose-6-phosphate dehydrogenase, and phosphoglycerate kinase genes, is the only recognizable human genetic material retained by the hybrids. Enclosure of the chromosomes in the lipid envelope was confirmed by electron and fluorescence microscopy and differential centrifugation. These lipochromosomes were fused with HGPRT(-) mouse cells (A9) in the presence or absence of polyethylene glycol and transferents were selected in hypoxanthine/aminopterin/thymidine (HAT) medium. The frequency of transfer was at least once per 10(5) cells, a minimum 10-fold improvement over previous methods. The selected cells contained HGPRT activity similar to the amount found in the A9/HRBC2 cells. Starch gel electrophoresis verified that the observed HGPRT activity in the transferents is due to the human enzyme. Human glucose-6-phosphate dehydrogenase and phosphoglycerate kinase were also identified electrophoretically in the transferents. Karyotyping with C and Q banding did not reveal the presence of the whole human X chromosome or a visible extra fragment of a human chromosome associated with the mouse genome. The biochemical data strongly suggest, however, that transfer of a portion of the human X chromosome has occurred in these transferents. Thus, at least three X-linked genes have been transferred from one cell to another with high frequency, using metaphase chromosomes.

Cell Line↗
PubMed1978

Biochemical heterogeneity in glutathione synthetase deficiency.

Two different clinical syndromes are associated with glutathione synthetase deficiency, one presenting with hemolytic anemia and 5-oxoprolinuria, the other with isolated hemolysis. We have differentiated these disorders on an enzymatic basis. In 5-oxoprolinuria, all cell types examined have grossly deficient enzyme activity and glutathione content. In contrast, in the nonoxoprolinuric variant, erythrocytes have decreased enzyme activity and glutathione content, whereas nucleated cells maintain substantial levels of both. The enzyme in this disorder is unstable in vitro and has shortened survival in intact erythrocytes. Nucleated cells appear able to maintain sufficient enzyme activity and concentrations of glutathione to suppress overproduction of 5-oxoproline.

Erythrocytes↗
PubMed1976

Normal amino acid uptake by cultured human fibroblasts does not require gamma-glutamyl transpeptidase.

The uptake kinetics for four amino acids (cystine, glutamine, methionine, and alanine) which are among the best gamma-glutamyl acceptors have been determined for normal human fibroblasts and for a cell line containing undetectable quantities (< 0.5% normal mean) of gamma-glutamyl transpeptidase activity. Apparent Km and V(max) for uptake for each of the four amino acids were normal in the mutant fibroblasts. Insulin increased the uptake of alpha-aminoisobutyrate as in control cells. levels of 16 amino acids were also normal in this cell strain; the intracellular concentrations of phenylalanine, cystine, and cysteine were increased. In human fibroblasts, amino acid transport appears to proceed normally in the absence of active gamma-glutamyl transpeptidase.

Amino Acids↗