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.

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.

Expression of human recombinant beta 2-glycoprotein I with anticardiolipin antibody cofactor activity.

To enable the synthesis of beta 2-glycoprotein I mutants we have established a stable Chinese hamster ovary cell line that expresses human beta 2-glycoprotein I up to 2.9 micrograms/10(6) cells/day. Recombinant beta 2-glycoprotein I is identical to the purified native protein with respect to cofactor activity revealed in a modified anti-cardiolipin ELISA. Autoimmune type anti-cardiolipin antibody requires recombinant beta 2-glycoprotein I in a dose-dependent manner to bind cardiolipin whilst binding of infectious type antibody is inhibited. The purified recombinant beta 2-glycoprotein I in serum free medium exists as two oligosaccharide species which upon deglycosylation have identical apparent molecular weight to the deglycosylated native protein.

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.

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.

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.

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.

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.

Increased permeability of psoriatic skin to the protein, plasminogen activator inhibitor 2.

The penetration and permeation of the recombinant protein plasminogen activator inhibitor type 2 (PAI-2) in two formulations, one containing a penetration enhancer, into the psoriatic and uninvolved skin of eight patients with plaque-type psoriasis were investigated. Penetration and permeation of PAI-2 were measured by gamma counting and imaging following radiolabelling of a fraction of the applied PAI-2 with (123)I. The feasibility of topical delivery of drug to psoriatic plaques was confirmed by the finding that the permeability of psoriatic plaques to radiolabelled PAI-2 (P=0.007) and free (123)I (P=0.001) was approximately tenfold higher than the permeability of uninvolved skin. The addition of a penetration enhancer improved the permeation of PAI-2 into psoriatic plaques from an average of 35% to 46% (P=0.005). Occlusion decreased the permeation amount of PAI-2 from 46% to 15% due to losses on the occlusive dressing (P=0.001).

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.

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.

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.

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.

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.

Detection of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2(PAI-2) in gingival crevicular fluid from healthy, gingivitis and periodontitis patients.

BACKGROUND: The regulation of plasminogen activation is a key element in controlling proteolytic events in the extracellular matrix. Our previous studies had demonstrated that in inflamed gingival tissues, tissue-type plasminogen activator (t-PA) is significantly increased in the extracellular matrix of the connective tissue and that interleukin 1beta(IL-1beta) can up regulate the level of t-PA and plasminogen activator inhibitor-2 (PAI-2) synthesis by human gingival fibroblasts. METHOD: In the present study, the levels of t-PA and PAI-2 in gingival crevicular fluid (GCF) were measured from healthy, gingivitis and periodontitis sites and compared before and after periodontal treatment. Crevicular fluid from106 periodontal sites in 33 patients were collected. 24 sites from 11 periodontitis patients received periodontal treatment after the first sample collection and post-treatment samples were collected 14 days after treatment. All samples were analyzed by enzyme-linked immunosorbent assay (ELISA) for t-PA and PAI-2. RESULTS: The results showed that significantly high levels of t-PA and PAI-2 in GCF were found in the gingivitis and periodontitis sites. Periodontal treatment led to significant decreases of PAI-2, but not t-PA, after 14 days. A significant positive linear correlation was found between t-PA and PAI-2 in GCF (r=0.80, p<0.01). In the healthy group, different sites from within the same subject showed little variation of t-PA and PAI-2 in GCF. However, the gingivitis and periodontitis sites showed large variation. These results suggest a good correlation between t-PA and PAI-2 with the severity of periodontal conditions. CONCLUSION: This study indicates that t-PA and PAI-2 may play a significant rôle in the periodontal tissue destruction and tissue remodeling and that t-PA and PAI-2 in GCF may be used as clinical markers to evaluate the periodontal diseases and assess treatment.

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.

Immunohistochemical demonstration of the plasminogen activator system in human gingival tissues and gingival fibroblasts.

The relative distribution of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) was studied in cultured human gingival fibroblasts, healthy gingival tissues and inflamed gingival tissues by immunohistochemistry. In cultured gingival fibroblasts t-PA, u-PA and PAI-1 were expressed in cytoplasm; u-PA and PAI-1 were more intensely stained than t-PA; PAI-2 was not detectable in gingival fibroblasts. Following interleukin 1 beta (IL-1 beta) stimulation, the intensity of intracellular staining for t-PA was increased and a number of cells staining strongly for PAI-2 were seen; no difference in the intensity of immunostaining level was noted for the expression of u-PA and PAI-1 between IL-1 beta stimulated cells and unstimulated cells. In healthy gingival tissues, u-PA and PAI-1 displayed a wide distribution throughout all the connective tissue and epithelium; t-PA localized mainly in the connective tissue while PAI-2 showed little association with the connective tissue but did faintly stain in the epithelial layer. In inflamed gingival tissues, staining for t-PA was significantly increased in the extracellular matrix of the connective tissue, whereas staining for u-PA, PAI-1 and PAI-2 was found to be slightly increased, but no significant difference was noted for staining when compared with the healthy gingival tissues. A granular distribution of t-PA, u-PA, PAI-1 and PAI-2 was noted around areas of inflammatory cell infiltration. These immunohistochemical findings indicate that the plasminogen activator system produced by fibroblasts may be influenced by the presence of the inflammatory mediator IL-1 beta. In addition, the significant increase of t-PA in inflamed connective tissue and the wide expression of these components around inflamed cells may contribute to connective tissue degradation and may relate to the migration and localization of monocytes/macrophages in inflamed tissue.

Effect of lipopolysaccharide from periodontal pathogens on the production of tissue plasminogen activator and plasminogen activator inhibitor 2 by human gingival fibroblasts.

Both tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) are important proteolysis factors present in inflamed human periodontal tissues. The aim of the present study was to investigate the effect of lipopolysaccharide (LPS) on the synthesis of t-PA and PAI-2 by human gingival fibroblasts (HGF). LPS from different periodontal pathogens including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum were extracted by the hot phenol water method. The levels of t-PA and PAI-2 secreted into the cell culture media were measured by enzyme-linked immunosorbent assays (ELISA). The mRNA for t-PA and PAI-2 were measured by RT-PCR. The results showed t-PA synthesis was increased in response to all types of LPS studied and PAI-2 level was increased by LPS from A. actinomycetemcomitans and F. nucleatum, but not P. gingivalis. When comparing the effects of LPS from non-periodontal bacteria (Escherichia coli and Salmonella enteritidis) with the LPS from periodontal pathogens, we found that the ratio of t-PA to PAI-2 was greater following exposure of the cells to LPS from periodontal pathogens. The highest ratio of t-PA to PAI-2 was found in those cells exposed to LPS from P. gingivalis. These results indicate that LPS derived from periodontal pathogens may cause unbalanced regulation of plasminogen activator and plasminogen activator inhibitor by HGF and such an effect may, in part, contribute to the destruction of periodontal connective tissue through dysregulated pericellular proteolysis.

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.

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.