Biomedical subjects
J H Buchanan
Publications and source records attributed to J H Buchanan.
Aminoglycoside antibiotic treatment of human fibroblasts: intracellular accumulation, molecular changes and the loss of ribosomal accuracy.
The aminoglycoside antibiotic, G418, was rapidly endocytosed by human fibroblasts. The endocytic uptake comprised two phases. A rapid phase in which G418 was first adsorbed to the cell surface, followed by a slower rate of uptake of 7 to 11 micrograms/mg cell protein/24 h. The rapid uptake was independent of temperature and a fluorescent derivative of G418 could be seen bound to the cell surface. This evidence suggests a nonspecific adsorption mechanism. The slower rate of accumulation may arise from fluid-phase pinocytosis. The effects of G418 and paromomycin on protein synthesis in vivo and in vitro were compared and it was concluded that G418 was more active on mammalian ribosomes. Finally, changes in the pattern of cellular proteins during treatment with either aminoglycoside were found only with G418. The changes in the pattern of proteins suggest the synthesis of stress proteins in treated cells. This change in pattern may be interpreted as a cellular response to abnormal protein synthesis arising from a loss of fidelity or some inhibitory function of G418 on membrane metabolism which may lead to a shortening of cellular life span.
Autofluorescence and ageing: changes in ribosome accuracy and lysosome [corrected] function.
Autofluorescence increased with decreasing proliferative capacity of fibroblast cultures. It also increased rapidly when cultures were treated with the aminoglycosides, streptomycin, paromomycin and G418, as well as with ammonium chloride. All these compounds are known to accumulate in lysosomes. Paromomycin is known to reduce proliferative capacity and G418 was found to have a similar effect. As these aminoglycosides accumulate in the lysosomes, promote errors in protein synthesis and reduce proliferative capacity this has led us to suggest that such compounds may cause a loss of homeostasis between erroneous protein synthesis and lysosome function. The increase in autofluorescence may be a consequence of reduced lysosome function arising from lack of control of the intralysosomal pH.
An island in a storm.
Explore the source record for details and available documents.
The accuracy of Q beta RNA translation. 1. Errors during the synthesis of Q beta proteins by intact Escherichia coli cells.
The fidelity of Q beta RNA translation by intact Escherichia coli cells has been studied. After infection, host protein synthesis was eliminated by adding rifampicin and the radioactive, phage-specified, proteins separated by one or two-dimensional gel electrophoresis. Labelled histidine and tryptophan were incorporated into the phage coat protein, whose message does not specify these amino acids, at a frequency of 0.09-0.13 per molecule. Errors leading to a change in the pI of the coat protein occurred at a rate of 0.05 per molecule, while the coat protein UGA stop codon was misread 6.5% of the time. These error rates are similar to data in some recent publications but much higher than the canonical 3-4 X 10(-4). They further provide a reference point in vivo to which the translation of the same message by E. coli extracts can be compared.
The accuracy of Q beta RNA translation. 2. Errors during the synthesis of Q beta proteins by cell-free Escherichia coli extracts.
The accuracy of Q beta translation by Escherichia coli extracts in polymix and a conventional Tris/Mg2+ system has been followed. Misinsertions of histidine and of tryptophan into the phage coat protein were less frequent in polymix than in Tris/Mg2+, as were errors leading to a change in the coat protein pI. Even the lowest Q beta error rates, however, were still an order of magnitude greater than those for poly(U) or poly(U-G) translation. Comparing Q beta translational errors made in vitro to those found in whole cells, histidine misinsertions were almost twice as frequent, errors leading to a coat protein charge change six times more frequent and tryptophan misinsertions at least 15 times more frequent in vitro. The relation of these findings to measurements of translational accuracy and to factors affecting fidelity is discussed.
The characterization of non-histone proteins whose amounts increase in chromatin from mouse hepatocarcinomas.
A fraction containing liver- and hepatoma-specific non-histone proteins has been isolated from the chromatin of mice. Amino acid analysis of this fraction shows that it contains 16 mol of glutamic acid, 10 mol aspartic acid, 7 mol of both arginine and lysine per 100 mol and contains no cysteine or tyrosine. The proteins in this fraction are strongly associated with DNA and are co-extracted with histones from chromatin with 0.25 M HCl. In chromatin from age-related hepatomas, the amount of this fraction increased six-fold. This increase in concentrations of these chromatin proteins may be associated with changes of chromatin structure necessary to initiate malignant growth in liver cells.
Intracellular accumulation of a fluorescent derivative of paromomycin in human fibroblasts.
Human fetal lung fibroblasts grown in the presence of dansyl-paromomycin (DNS-Pm), a fluorescent derivative of the aminoglycoside antibiotic, paromomycin, probably accumulate DNS-Pm in the lysosomes. The intracellular concentration of DNS-Pm is proportional to the extracellular concentration and to the length of time cells are exposed to the compound. The accumulation of DNS-Pm by human fibroblasts continued to increase for several days, reaching a saturation after 7 days. The kinetic data are consistent with the establishment of a steady state in the cell between fluid-phase pinocytosis and exocytosis of DNS-Pm. About 80% of the intracellular DNS-Pm ws released in 24 hr when fresh medium without the analogue was added. The residual 20% remained within the cells, suggesting that it may be irreversibly bound to the lysosomes, endoplasmic reticulum, or ribosomes. The uptake of paromomycin by cells in culture may be a useful means to study error propagation during growth and lifespan of cells in vitro.
Autofluorescence as an index of ageing in human fibroblasts in culture.
An exponential increase in autofluorescence (AF) during serial passaging of human diploid fibroblasts, strain MRC-5, was observed using a fluorescence-activated cell sorter. Skin fibroblasts from patients with premature-ageing syndromes had high levels of AF, whereas virus-transformed cell lines had negligible amounts of AF.
Optimum conditions for growth and longevity of chick embryo fibroblasts in culture.
Chick embryo fibroblasts are often used as a model of cellular ageing in vitro. It seems important to culture these cells in optimum conditions which may not be the same as those for mammalian cells. An incubation temperature of 40 degrees C and the use of chicken serum were found to be optimum for growth and longevity of chick embryo fibroblasts in culture.
Accuracy of in vitro protein synthesis: translation of polyuridylic acid by cell-free extracts of human fibroblasts.
Errors in translation have been measured in cell-free protein synthesising extracts derived from cultured MRC-5 human diploid fibroblasts of limited lifespan. Using polyuridylic acid as messenger, the error frequency for the misincorporation of leucine was 1-2%. It was found that varying the concentration of leucine increased the error frequency for leucine misincorporation. No difference could be detected in the accuracy of translation with increasing cell age, from passage 25 to 55. The aminoglycoside antibiotic, paromomycin, was shown to have a profound effect on the leucine misincorporation, increasing the error frequency of this amino acid twenty-to fortyfold. However, there was no difference in the paromomycin-induced errors wtih increasing cell age. Another effect of this antibiotic is that it inhibits the incorporation of the cognate amino acid phenylalanine. It was found that passage 55 cell extracts were less inhibited by paromomycin than similar extracts made from lower passage cells (passages 25 and 40). When the accuracy of translation of cell extracts made from human transformed cells (HeLa) and untransformed cells (MRC-5) was compared, no detectable difference could be found. Paromomycin increased the leucine errors in extracts made from HeLa cells to a similar degree to that observed for MRC-5 fibroblasts.
Evidence for increased proteolytic activity in ageing human fibroblasts.
We have shown that ultrastructural changes accompany ageing in the human fetal lung fibroblast strain MRC-5. In order to further characterise these changes a method has been devised to prepare subcellular fractions from MRC-5 fibroblasts using a discontinous gradient of metrizamide preceded by a low-speed centrifugation. An investigation was carried out to compare subcellular fractions prepared from early-passage and senescent cells. Proteolytic activity associated with lysosomes and soluble proteolytic activity were found to be increased in senescent cells.
Studies on phosphoglucose isomerase from cultured human fibroblasts: absence of detectable ageing effects on the enzyme.
Some properties of the enzyme phosphoglucose isomerase (PGI) from the foetal lung fibroblast strain MRC-5 have been investigated throughout the in vitro lifespan of this cell strain. No significant age-related alterations in specific activity or thermostability of PGI could be detected. Titration of enzymatic activity with antibody directed against purified PGI showed no detectable differences in PGI from extracts of early passage cells compared with enzyme from senescent cells. The effect of p-fluorophenylalanine incorporation on PGI was examined in early passage fibroblasts. Thermostability studies showed increased heat lability of PGI from analogue treated cells when compared with enzyme from control cells at the same passage. However, no inactive PGI protein could be detected by anti-serum titration in extracts from analogue-treated cells. The results indicate that no significant amount of altered or inactive PGI is produced in ageing fibroblasts.
Fidelity of histone synthesis in cultured human fibroblasts.
The results in the literature to support Orgel's general error hypothesis of ageing only provide indirect evidence that errors in protein synthesis increase during senescence. This study attempts to provide direct evidence of errors in protein synthesis by measuring the misincorporation of 35S-methionine into histone H1 obtained from young and old fibroblasts (MRC-5). The conclusions that can be drawn from this study are: (a) the error level for the misincorporation of methionine into histone H1 is less than 7 methionines/10(5) amino acids and 2-3 methionines/10(4) amino acids in young and old cells respectively; (b) a methionine containing fraction associated with H1 is obtained after the final purification. The amount of this fraction increases with the age of the cell culture as does the number of methionine residues; (c) there is a variation in the complexity of H1 polypeptide chains, the complexity increasing with the age of cultured cells.
A cystine-rich protein fraction from oxidized alpha-keratin.
A soluble fraction of alpha-keratin was obtained on fission of disulphide bonds. The fraction was soluble in the oxidizing solution and would normally be lost when such procedures are used for isolating keratose fractions. This fraction, which constituted 6% by weight of keratin, was rich in cystine, and about 30% of the fraction had a mol.wt. of less than 20 000.
Use of a programmable pocket calculator in processing amino acid analysis data.
Explore the source record for details and available documents.
Increased degradation rates of protein in aging human fibroblasts and in cells treated with an amino acid analog.
Explore the source record for details and available documents.