Biological and clinical aspects of plasminogen activator inhibitor type 2.
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Apoptosis↗
Biomedical subjects
C L Bunn
Publications and source records attributed to C L Bunn.
Decreased accuracy of protein synthesis in extracts from aging human diploid fibroblasts.
The accuracy of protein synthesis has been measured in extracts from human diploid fibroblasts of different ages. Extracts were supplied with purified mRNA for the coat protein of the cowpea variant of tobacco mosaic virus (CcTMV), which lacks codons for cysteine and methionine. The presence of 35S-cysteine in CcTMV coat protein synthesized during translation reactions therefore represents translational error. Translation reactions were performed with extracts from young fibroblasts (less than 50% of life span completed) and old fibroblasts (more than 90% of life span completed), and the translation products were purified by immunoprecipitation and analyzed by polyacrylamide gel electrophoresis. The error frequency increased from 4.2 x 10(-5) cysteines/amino acid in young cell extracts to 2.9 x 10(-4) cysteines/amino acid in old cell extracts. Cysteine incorporation was not due to nonspecific binding, and could be increased approximately sixfold by the addition of the misreading antibiotic, paromomycin. It is concluded that translational accuracy is not stable during aging in vitro, and it is proposed that this decrease in the fidelity of information transfer could be responsible for the variety of changes observed in aging cultured human cells.
Amino Acids↗
Altered sensitivity of protein synthesis to paromomycin in extracts from aging human diploid fibroblasts.
Age-related differences in the effects of paromomycin (Pm) on protein synthesis have been investigated in translation reactions with extracts derived from young and old human diploid fibroblasts. Translation products from reactions directed by endogenous or exogenous mRNA were analyzed by polyacrylamide gel electrophoresis and fluorography. The exogenous mRNA lacked codons for cysteine, and therefore cysteine incorporation into translation products represented translational error. This laboratory has previously used this assay to show that the basal translational error level in the absence of Pm increases in extracts from old fibroblasts. In this report, Pm stimulated the misincorporation of cysteine by 6-7 fold over cysteine misincorporation levels in the absence of Pm. This degree of Pm stimulation was similar in extracts from young and old fibroblasts. However, other results showed quantitative differences in the responses to Pm between young and old cell extracts. Old cell extracts were less sensitive to the stimulation of the rate of protein synthesis, and more sensitive to the inhibition of protein synthesis, by Pm. It is proposed that aging human diploid fibroblasts contain altered ribosomes which react differently with Pm.
Aging↗
The experimental manipulation of keratin expression and organization in epithelial cells and somatic cell hybrids.
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Animals↗
Inherent resistance of HeLa cell derivatives to paromomycin.
The human tumor-derived cell line HeLa S3 and nuclear and mitochondrial gene mutants derived from it are resistant to the aminoglycoside antibiotic, paromomycin (PAR). Other carcinoma-derived cells, SV40-transformed cells, and four human diploid fibroblast cell lines are all sensitive to PAR. Sensitivity is dependent on cell density, and at cell numbers greater than 400/cm2 sensitive cells will proliferate in PAR. The resistance to PAR is inherited in a dominant manner in cell-to-cell fusion hybrids, but is not transferred in cytoplast-to-cell fusions. PAR resistance is therefore encoded by a nuclear gene(s). Resistance to PAR is not caused by changes in the response of mitochondrial or cytoplasmic protein synthesis to PAR in vitro. The uptake of PAR is similar in resistant and sensitive cells, and dimethyl sulfoxide does not render resistant cells more sensitive. Thus, HeLa cell PAR resistance is unlike previously reported ribosomal mutations and may derive from differences in the intracellular metabolism of PAR.
Adrenal Cortex Neoplasms↗
The accuracy of protein synthesis in reticulocyte and HeLa cell lysates.
The accuracy of translation in protein synthesis is measured as the rate of misincorporation of a particular amino acid, different from that specified by an mRNA codon, into protein. The cowpea variant of tobacco mosaic virus, CcTMV, contains no cysteine or methionine in its coat protein. Translation in vitro of purified CcTMV coat protein mRNA by rabbit reticulocyte and HeLa cell lysates has been performed. The coat protein product was purified by immunoprecipitation with specific antisera, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The error rate was measured by comparing the incorporation of [35S]cysteine with incorporation of [3H]leucine, and the total CcTMV coat protein synthesized was calculated from its known leucine content. An error rate of (1-2) X 10(-3) cysteines/CcTMV coat protein was obtained with reticulocyte lysates. If errors were cysteine incorporation in place of arginine, this number is converted to 3 X 10(-4) cysteine/codon. If cysteine was incorporated anywhere in the polypeptide, the rate is 9 X 10(-6) cysteines/amino acid. The error frequencies with HeLa cell lysates were 6-fold higher. Paromomycin, a eukaryotic misreading antibiotic, increased error rates 10-fold in both lysates. These data compare well with in vivo measurements and suggest that some transformed cells may survive with higher mistranslation rates.
Animals↗
Restriction enzyme analysis of mitochondrial DNA in aging human cells.
Human diploid fibroblasts show a limited lifespan in vitro. To investigate the integrity of mitochondrial DNA (mtDNA) in aging fibroblasts, whole cell DNA samples from the human cell line IMR-90 have been prepared at 36, 22, and 3 population doublings (PD) from the end of the lifespan (63 PD). These DNA samples were then digested separately with 19 different restriction endonucleases, and the resulting fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose filters. Fragment sizes were revealed by hybridization to 32P-labelled mouse mtDNA and autoradiography, and were compared with computer maps of fragments generated from the known sequence of human mtDNA. These 19 enzymes recognize a total of 297 recognition sites comprising 1315 nucleotide base pairs (bp), approximately 8% of the human mtDNA (16 569 bp). Control experiments reveal that a minor component representing as little as 5% of the total mtDNA can be detected. No changes were seen in the restriction fragment pattern with fibroblast cell age. It is concluded that there are no large deletions, insertions, or rearrangements in human mtDNA, and no single base changes in the detectable regions. This suggests efficient maintenance of mtDNA molecules and/or elimination of damaged mtDNA during fibroblast cell lifespan.
Base Composition↗
Intermediate filament expression and lifespan potential in human somatic cell hybrids.
Limited lifespan human diploid fibroblast cells have been fused with the HeLa derived cell line HEB 7A which possesses transformed growth characteristics and unlimited division potential. HEB 7A expresses keratin intermediate filaments, while the fibroblast cells express only vimentin intermediate filaments. Independently arising clones of hybrids were examined for the presence of keratin by indirect immunofluorescence. Of 11 limited lifespan hybrids, all were keratin negative and possessed the growth characteristics of the fibroblast parent. Of 8 transformed hybrids, 6 arising early after fusion and 2 arising late, all were keratin-positive and simultaneously expressed the transformed growth characteristics of loss of density dependent growth inhibition, low serum dependence, and anchorage independence. It is concluded that the growth properties of these hybrids are associated with the type of intermediate filament expressed. The intermediate filament expression is therefore a marker of proliferative potential in these hybrids.
Cell Division↗
Clonal variations in keratin. Intermediate filament expression by human somatic cell hybrids.
The intermediate filament composition of differentiated vertebrate cells provides a stable phenotype which appears to be specifically regulated in each cell type. In order to analyse the regulation of intermediate filament expression we have constructed human somatic cell hybrids from the fusion of the HeLa-derived cell line HEB7A and a normal human diploid fibroblast, GM2291. These parental cells differ with respect to the presence or absence of keratin intermediate filaments. Isolation of independently arising clones produced two classes of hybrids. One class expresses keratin in a stable manner and the other class lacks keratin altogether. Indirect immunofluorescence of hybrid cells using antikeratin antiserum demonstrates that there are variations in the intensity and organization of cytoskeletal keratin staining. SDS-PAGE comparisons of cell extracts from these hybrids indicates that there are quantitative differences in the relative amounts of individual keratin polypeptides as well. These clonal variations have allowed us to begin assessing the consequences of genetic interactions between cell types that are normally capable of closely regulating different subsets of intermediate filament genes.
Cell Fusion↗
Monitoring asthma with a Mini-Wright Peak Flow Meter.
Clinical assessments of the degree of airway obstruction in asthma are known to be unreliable. Objective measurements of pulmonary function are essential to assessing the severity of asthma. Recently, an inexpensive, portable machine, the Mini-Wright Peak Flow Meter, has become available for clinic and home use. This article describes ways the nurse practitioner can use this meter to improve diagnosis and treatment of asthma. Peak flow measurements can be used to diagnose asthma, and are especially useful in atypical and severe presentations of the disease. Treatment is enhanced by both clinic and home monitoring of response to medications. Regular home monitoring can also improve patients' abilities to provide self-care. Several examples from a case study are given which illustrate the usefulness of home monitoring in medication management, in detection of diurnal variation of the asthma and in the identification of acute illness.
Adolescent↗
Segregation of mitochondrial DNA in human somatic cell hybrids.
The maintenance of mtDNA has been examined in human intraspecific hybrid cells constructed from the fusion of HEB7A, a HeLa tumor cell line carrying the mitochondrially coded chloramphenical (CAP) resistance mutation, and GM 2291, a limited lifespan human diploid fibroblast which is CAP sensitive. These two cells can be distinguished by a polymorphism in a site for the restriction endonuclease, HaeIII. Independently isolated clones of hybrid cells were characterized for their growth properties (either normal limited lifespan or transformed and "immortal"). Whole cell DNA preparations were made from each hybrid, digested with HaeIII, and the resultant fragments were detected by hybridization to 32P labelled mouse mtDNA as probe. Experiments with mixtures of HEB7A and GM 2291 DNA reveal that HEB7A mtDNA can be detected when it constitutes as little as 5% of the total cell mtDNA. The results indicate that the HEB7A mtDNA is lost from most hybrids, and when it does persist it is usually a minor component of total mtDNA. The addition of CAP at the time of fusion slightly increases the quantity of HEB7A mtDNA, but not enough to confer CAP resistance. Furthermore, five limited lifespan hybrids contained no detectable HEB7A mtDNA, while three transformed hybrids contained varying quantities of HEB7A mtDNA, suggesting that retention of this tumor form of mtDNA is associated with tumor growth behavior. These results suggest that cytoplasmic genetic incompatibility occurs in intraspecific hybrids.
Chloramphenicol↗
Limited lifespan in somatic cell hybrids and cybrids.
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Cell Division↗
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.
Cell Survival↗
Cybrid formation in mouse L cells: the influence of cytoplast-to-cell ratio.
The frequency of cybrid colony formation was measured in fusions between enucleated chloramphenicol (CAP)-resistant mouse cells and CAP-sensitive mouse cells in varying ratios. By labeling the CAP-resistant cytoplasts with polystyrene beads and then performing the same fusions with CAP-sensitive cells, the frequency of cybrid fusions could be measured. Comparison of the frequency of viable cybrids (cybrid colonies) with the frequency of cybrid fusions showed that, with increasing fusion ratios of cytoplasts to cells, the proportion of cells fused to cytoplasts increased. Further, the viability of cybrid fusions increased from about 1 in 500 to nearly 1 in 60 over the range of cytoplast-to-cell ratios studied.
Cell Fusion↗
Carbomycin resistance in mouse L cells.
A mutant has been isolated from the mouse cell line LM(TK-) which is stably resistant to the macrolide antibiotic, carbomycin. Mitochondrial protein synthesis in this mutant was carbomycin resistant and chloramphenicol sensitive. Fusions between carbomycin-resistant and -sensitive cells produced hybrids, most of which were sensitive to 10 microgram/ml carbomycin. At 7.5 microgram carbomycin/ml, the average population resistance is low initially but increases with time. Carbomycin-resistant cells were enucleated and fused with carbomycin-sensitive cells under a variety of selective regimes designed to allow growth of carbomycin-resistant cytoplasmic hybrids (cybrids). No transfer of carbomycin resistance via the cytoplasm was detected. Karyoplasts from carbomycin-resistant cells showed a low transfer of resistance to 7.5 microgram carbomycin/ml in karyoplast-cell fusions. Carbomycin resistance in this mutant is therefore most likely encoded in a nuclear gene.
Cell Nucleus↗
Mitotic segregation of cytoplasmic determinants for chloramphenicol resistance in mammalian cells. I: Fusion with mouse cell lines.
The segregation of cytoplasmically inherited chloramphenicol (CAP) resistance in mouse cells was investigated in fusions between CAP-resistant cells or cytoplasts (enucleated cells) and CAP-sensitive cells of varying tissue origin. All hybrids formed in cell-cell fusions were initially CAP-resistant, indicating that CAP resistance is dominant. Hybrids from fusions of cells of the same tissue origin (homologous) were stably CAP-resistant, whereas the hybrid population from fusions of different origins (heterologous) showed a rapid diminution of average CAP resistance. Individual hybrid clones from these heterologous fusions also showed an overall loss of CAP resistance, and a wide variation in CAP resistance which is consistent with a large number of genetic determinants (possibly mitochondrial DNA molecules) contributing to the CAP phenotype. Similar results were obtained from cytoplast-cell fusions, so the observed CAP segregation is not the result of nuclear-nuclear interactions. This segregation of CAP resistance constitutes a second criterion of cytoplasmic inheritance in mammalian cells.
Animals↗
Mitotic segregation of cytoplasmic determinants for chloramphenicol resistance in mammalian cells II: Fusions with human cell lines.
Cytoplasmically inherited chloramphenicol (CAP) resistance in human cells has been used to study the interaction between sensitive and resistant mitochondria. Cybrids between two HeLa cells were stable for resistance, grew rapidly and cloned well in CAP, and were O2 tolerant. HeLa-HeLa hybrids were also stable up to 70 doublings in the absence of CAP. Cybrids between HeLa and WI-L2 cells were unstable for resistance for up to 40 doublings, grew slowly and cloned poorly in CAP, and were O2 sensitive (S phase). The growth rate then increased and the cells became stable for resistance, cloned well, and were not O2 sensitive (F phase). Doubling time for S but not F phase cells was proportional to CAP concentration, indicating that both kinds of mitochondria were present and functioning. The instability of CAP resistance in many interstrain but not in intrastrain mouse and human cybrids and hybrids is interpreted in relation to lower eukaryotes.
Cell Cycle↗
Cytoplasmic inheritance in mammalian tissue culture cells.
A series of intraspecific, interspecific and interorder somatic cell cybrids and hybrids have been prepared by fusions in which one of the parents contained the cytoplasmically inherited marker for chloramphenicol (CAP) resistance. A clear relationship has been established between the expression of the CAP-resistant (CAP-R) determinants in the fusion products and the genetic homology of the parents. With increased genetic divergence, the acceptability of the CAP-R mitochondria decreased. Intraspecific cybrids and hybrids of the same strain were stable for the CAP-R marker, while those between strains were stable only in CAP. Intergeneric mouse-hamster cybrids occurred at a high frequency but were unstable in CAP, while CAP suppressed hybrid formation 100-fold. Interorder cybrids (CAP-R human X CAP-S mouse) occurred either at a moderate frequency and were stable at a low frequency and were unstable in CAP. Interorder hybrids could only be formed by challenging HAT-selected hybrids with CAP or by direct selection in ouabain and CAP. Reciprocal interorder crosses between CAP-R mouse and CAP-S human cells were unsuccessful. Interspecific cybrids contain only the chromosomes of the CAP-S parent. Interspecific hybrids selected directly in CAP segregated the chromosomes of the CAP-S parent, while hybrids selected in HAT and then CAP segregated those of the CAP-R parent. The mitochondrial DNA(mtDNA) of all mouse-human cybrids and most HAT and then CAP-selected hybrids contain only the mtDNA of the CAP-S mouse parent. However, preliminary evidence suggests that one of these hybrids contains both mouse and human mtDNA sequences.
Animals↗