Reversible ultrastructural changes in human fibroblasts grown in hepes buffered MCDB-104 supplemented with human serum

Verdery, Roy B.; Nist, Cynthia; Wight, Thomas N.; Glomset, John A.

doi:10.1007/bf02618420

Cited by 12 publications

(1 citation statement)

References 14 publications

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“…With regard to serum, it is interesting to note that degradation in a line of human skin fibroblasts is 13% when serum is included in the labeling medium and 23% when it is omitted (Steinmann et al, 1981); clearly, biological differences between cell lines may account for some of the variation in reported values of degradation. Although changes in pH had no direct effect on intracellular degradation, the small but significant difference associated with use of organic buffers demonstrates that these compounds have other effects on cell metabolism besides regulation of hydrogen ion concentration (Verdery et al, 1981). There has been only one other report of a decrease in degradation below the control level: Gallagher et al (1982) found that dichloromethylenebiphosphonate lowers degradation in cultured bone cells.…”

Section: Extent Of Degradationmentioning

confidence: 99%

Collagen degradation in human lung fibroblasts: Extent of degradation, role of lysosomal proteases, and evaluation of an alternate hypothesis

Bienkowski¹

1984

Journal Cellular Physiology

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Experiments were conducted to determine the extent and variability of collagen degradation in human fetal lung fibroblasts. Cells were incubated with [14C]proline, and degradation was measured by determining the hydroxy[14C]proline in a low molecular weight fraction relative to total hydroxy[14C]proline. Average (basal) degradation in stationary phase HFL-1 cells incubated for 8 h was 16 +/- 3%, and substantial alterations in the composition of the labeling medium, e.g., omitting serum and varying pH between 6.8 and 7.8, had no effect. Organic buffers slightly lowered degradation in a manner that was independent of pH. Collagen degradation in two other lung cell lines, Wl-38 and lMR-90, did not differ from the level in HFL-1. Degradation was significantly higher (23 +/- 5%) in HFL-1 cultures labeled for 24 h rather than 8 h, and pulse-washout experiments showed that the rate of degradation was not uniform: after an 8-h pulse, 11% of the hydroxy [14C]proline in the medium was in the low molecular weight fraction, but 31% was in this fraction after a 16-h washout. The lack of effect of either serum deprivation or elevated pH suggests that lysosomal proteases have no direct role in basal degradation; however, NH4Cl decreased the enhanced degradation observed in ascorbate deficiency to basal level, indicating that abnormal molecules synthesized under those conditions are degraded by lysosomal proteases. The appearance of small hydroxy[14C]proline-containing molecules was inhibited by alpha alpha'dipyridyl and cycloheximide in a dose-dependent and reversible manner, demonstrating that their production depends on enzymatic hydroxylation of proline and protein synthesis.

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Section: Extent Of Degradationmentioning

confidence: 99%

Collagen degradation in human lung fibroblasts: Extent of degradation, role of lysosomal proteases, and evaluation of an alternate hypothesis

Bienkowski¹

1984

Journal Cellular Physiology

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Cultured amniotic fluid cells for prenatal diagnosis of lysosomal storage disorders: A methodological study

Arnon

Ornoy

Bach³

1986

Prenatal Diagnosis

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The influence of culture conditions on the ultrastructure and enzyme activities of amniotic fluid cells are reported. Morphological changes were determined as a function of the number of lysosomal-like inclusion bodies per cell, and these results correlated to the activity of beta-hexosaminidase, alpha-mannosidase, beta-glucuronidase, arylsulphatase C and 5' nucleotidase. The parameters examined were pH of the culture media, type of media, increasing cell passage and day of harvest. Our results indicate that enzyme activities are less sensitive to changes in culture conditions as compared to ultrastructural changes. We therefore recommend that in order to obtain reliable ultrastructural results for the diagnosis of storage disorders, cultures should be grown in MEM as the culture medium, the pH of the medium carefully monitored to remain below pH 7.4, examining the cultures no later than the eighth cell passage and no later than the 10th day after subculture.

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Hepes may stimulate cultured endothelial cells to make growth-retarding oxygen metabolites

Bowman

Berger

Butler

et al. 1985

In Vitro Cell Dev Biol

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Zwitterion buffers are often used to modulate the pH of cell culture medium but their effect on cultured cells is controversial. We found that addition of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) caused superoxide dismutase (SOD) inhibitable increases in nitroblue tetrazolium dye reduction and SOD and catalase inhibitable decreases in the growth of cultured bovine pulmonary artery endothelial cells. The findings suggest that HEPES stimulates endothelial cells to make toxic oxygen metabolites that contribute to decreased cell growth.

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Reversible ultrastructural changes in human fibroblasts grown in hepes buffered MCDB-104 supplemented with human serum

Cited by 12 publications

References 14 publications

Collagen degradation in human lung fibroblasts: Extent of degradation, role of lysosomal proteases, and evaluation of an alternate hypothesis

Collagen degradation in human lung fibroblasts: Extent of degradation, role of lysosomal proteases, and evaluation of an alternate hypothesis

Cultured amniotic fluid cells for prenatal diagnosis of lysosomal storage disorders: A methodological study

Hepes may stimulate cultured endothelial cells to make growth-retarding oxygen metabolites

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