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Biomedical subjects

C L Bunn

Publications and source records attributed to C L Bunn.

31 records · Page 2Linked to original sources
PubMed1980

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↗
PubMed1977

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↗
PubMed1977

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↗
PubMed1977

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↗
PubMed1977

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↗
PubMed1976

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↗
PubMed1975

Cytoplasmic transfer of chloramphenicol resistance in human tissue culture cells.

The cytoplasmic inheritance of human chloramphenicol (cap) resistance has been demonstrated by removing the nuclei of cells of the CAP-resistant HeLa strain 296-1 (enucleation) and fusing them to a CAP-sensitive HeLa strain lacking nuclear thymidine kinase. Plating the fusion products in bromodeoxyuridine and CAP resulted in the growth of about 150 colonies/10(6) parent cells plated. Permanent cell lines (cybrids) grown from such fusions have been designated HEB. A recloned HEB cybrid (HEB7A) has also been enucleated and fused to hypoxanthine phosphoribosyl transferase (HPRT)-deficient HeLa cells (S3AG1) and HPRT-deficient lymphocytes (WAL-2A). Cybrids were selected in thioguanine and CAP. In the fusion of enucleated (en) HEB7A to S3AG1, 1,200 colonies/10(6) parents were observed. Fusion of enHEB7A to WAL-2A was done in mass culture and cybrids were obtained on three separate occasions. In every case the parental controls were negative. All isolates tested from the above fusions have the CAP-resistant characteristics, in vivo and in vitro, of the enucleated parent and the nuclear characteristics of the CAP-sensitive parent, such as chromosome number, morphology, and specific isozyme and chromosome markers. Therefore, it can be concluded that CAP resistance is coded in the cytoplasm and not in the nucleus of 296-1 cells. Furthermore, this resistance can be transferred to cells of widely different origin and differentiated state. These studies represent the first genetic evidence of cytoplasmic inheritance in human cells.

Cell Division↗
PubMed1974

Cytoplasmic inheritance of chloramphenicol resistance in mouse tissue culture cells.

A chloramphenicol-resistant mutant, isolated from mouse A9 cells, was enucleated and fused with a nucleated chloramphenicol-sensitive mouse cell line. Resultant fusion products, cytoplasmic hybrids (or "cybrids"), were selected as resistant to chloramphenicol, and had the nuclear markers and chromosome complement of the chloramphenicol-sensitive parent. These cybrids appeared at the high frequency of 2-8 per 10(4) cells plated. Neither parent produced any colonies when plated under identical selective conditions. Fusion between enucleated chloramphenicol-sensitive cell fragments and the chloramphenicol-sensitive cell produced no resistant colonies, suggesting that chloramphenicol resistance is not due to an increase in the ratio of cytoplasm to nucleus. Furthermore, fusions between resistant and sensitive nucleated cells produced resistant hybrids at a frequency 100 times less than that of resistant cybrids. Thus, these stable chloramphenicol-resistant cybrids result from the fusion of a chloramphenicol-resistant cytoplasm with a chloramphenicol-sensitive cell. It is proposed, therefore, that chloramphenicol resistance is a cytoplasmically inherited characteristic in this mouse cell line.

Animals↗
PubMed1970

Biogenesis of mitochondria. 18. A new class of cytoplasmically determined antibiotic resistant mutants in Saccharomyces cerevisiae.

New mutant yeasts resistant to the antibiotics chloramphenicol and mikamycin were isolated. They are mitochondrial mutants, characterized by several criteria as cytoplasmically determined. Biochemical studies show that amino acid incorporation into protein in vitro by mitochondria isolated from cells resistant or sensitive to mikamycin or chloramphenicol is inhibited by these antibiotics. Although aerobically-grown resistant strains of Saccharomyces cerevisiae are not affected by mikamycin or chloramphenicol, it is found that the mitochondrial protein-synthesizing system of anaerobically grown cells is inhibited in vivo. Cross resistance among the antibiotics chloramphenicol, mikamycin, erythromycin, lincomycin, carbomycin, and spiramycin is reported. All erythromycin resistant mutants, unlike the others, are resistant to erythromycin in vivo and in vitro. The results indicate that some of the cytoplasmic mutations (mikamycin and chloramphenicol resistance) are expressed at the mitochondrial membrane, whereas others (erythromycin resistance) possibly reflect changes in mitochondrial ribosomal proteins. We further suggest that conformational changes, either in the membranes or ribosomes, are likely to account for the observed antibiotic cross resistances.

Anti-Bacterial Agents↗
PubMedDate not supplied

Metallothionein expression and stress responses in aging human diploid fibroblasts.

Metallothioneins (MTs) are low molecular weight proteins with a high cysteine content that are inducible by heavy metals and by other conditions of environmental stress. This laboratory was investigated in human diploid fibroblasts the induction of MTs by cadmium and by dexamethasone, and the induction of heat shock proteins, as models for age-related changes in gene expression that reflect the ability of old cells to respond to environmental stress. Old cells were more sensitive to the toxic effects of CdCl2 in the concentration range 100-175 microM. Analysis of 35S-cysteine-labelled cell extracts by polyacrylamide gel electrophoresis and fluorography showed that in the absence of any inducer, old cells have a 3.7-fold increase over young cells in the basal level of MT. The rate and extent of induction of MT by CdCl2 was reduced in old cells: Exposure of old cells to 100 microM CdCl2 for 18 h resulted in MT levels about 33% of the amount in young cells. Northern blot analysis showed that the changes in MT protein levels occurred in parallel with changes in mRNA levels, which implicates transcriptional control as the origin of these aging changes. These young/old differences in MT synthesis were maintained in density-arrested cultures, indicating that the aging changes were not due to differences in the cell cycle status of these cell populations. The rate and extent of induction of a 68-kDa heat shock protein were also reduced in old cells, which showed an increase in basal, uninduced level of this protein similar to MT. In contrast, old cells retained the ability to synthesize MTs in response to dexamethasone at a rate similar to that in young cells.

Blotting, Northern↗