MRC-5 Human Fibroblasts Research Articles

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 with 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.

Stimulation of cellular thymidine kinases by human cytomegalovirus.

Thymidine kinase (TK) activity in WI-38 and MRC-5 human fibroblasts was analyzed by discontinuous polyacrylamide gel electrophoresis (disc-PAGE) and discontinuous glycerol gradient electrophoresis (disc-GEP) after subculture or human cytomegalovirus (HCMV) infection. Two peaks of TK activity with different relative fraction-of-migration (R(f)) values were resolved by disc-PAGE or disc-GEP in extracts from log-phase and infected cells. Growing WI-38 cells expressed a slowly migrating (R(f) = 0.14 PAGE, R(f) = 0.4 GEP) peak of TK activity, which was partially inhibited by 1.0 mM dCTP, but which retained little activity at pH 4.5. Growing MRC cells also displayed a slowly migrating peak (R(f) = 0.10 PAGE) with similar properties. Both cell types expressed a faster-migrating TK activity (R(f) = 0.45 PAGE, R(f) = 0.7 GEP) in the growing and resting state that was strongly inhibited by 1 mM dCTP but retained 50% activity at pH 4.5. When either cell type was infected with HCMV, there was a rapid and high-level stimulation of the slowly migrating form of TK and a slight stimulation of the faster-migrating form. Two strains of HCMV (AD169 and Town) failed to produce an electrophoretically distinct virus TK in either cell type after infection. TK enzymes were partially purified by disc-GEP from extracts of log-phase WI-38 or AD169-infected WI-38 cells. Characterization of these enzymes with respect to phosphate donor specificity, pH optima, thermostability, and salt inhibition showed the HCMV-stimulated TKs to be of cellular origin.

Chromosome changes during the in vitro ageing of MRC-5 human fibroblasts

Summary The frequency of polyploidy, aneuploidy and chromosome abnormalities was recorded at intervals throughout the lifespan of fetal lung fibroblasts strain MRC-5. The frequency of polyploidy rose significantly at passage 50, which is close to the beginning of the senescent phase, and a further increase was seen at passage 60, which is near the end of the lifespan. Aneuploidy and chromosome abnormalities increase significantly only at passage 60. The effect of incubation at 32, 34 and 40°C was also examined. Senescence is accelerated at 40°C, and this is accompanied by a dramatic rise in chromosome abnormalities and polyploidy. No similar effect is seen at the lower temperatures. The overall results suggest that chromosome changes are a secondary consequence of the cellular deterioration which leads to ageing, rather than being a major cause of ageing.

Sunlight and moonlight

Composite cements based on α-tricalcium phosphate (α-TCP) and nanostructured fluorapatite (FAp), α-TCP/FAp, appropriate for dental usage as root canal filling material, have been prepared with the improved mechanical properties and biocompatibility. The influence of FAp concentration on the composite cement properties was investigated. The processing conditions for FAp powders synthesis were optimized and the powder with optimal properties was chosen to obtain α-TCP/FAp composite types of cement. α-TCP powder was prepared by calcination of hydrothermally synthesized hydroxyapatite (HAp) at 1500 °C for 2 h. Morphology and phase composition of powders were investigated by X-ray diffraction (XRD), Fourier transformed infrared spectroscopy (FTIR), Field emission scanning electron microscopy (FESEM), and energy dispersive X-ray spectroscopy (EDS). Soaking of α-TCP/FAp specimens in simulated body fluid (SBF) affected the successful transformation of α-TCP into HAp, while the compressive test revealed the influence of this transformation and FAp content on the mechanical properties of the composites. Specimen containing 5% of FAp after 10 days in SBF showed the highest compressive strength. In vitro MTT and DET biocompatibility tests, performed in the culture of MRC-5 human fibroblast cells, revealed that α-TCP/FAp had no cytotoxic effect. These initial findings showed that α-TCP/FAp composite cements could yield favorable mechanical properties with no adverse effect on biocompatibility.