Age related and lectin influenced changes of exoglycosidases activity in cultured chick embryonic skin fibroblasts

beta-N-acetyl-D-glucosaminidase, beta-N-acetyl-galactosaminidase and a beta-D-galactosidase activity was determined in untreated and lectins (ConA, PNA, SBA and WGA) treated chick embryonic skin fibroblasts at two incubation stages. Activity of all three glycosidases increased between 7 and 14 incubation days. ConA and WGA affected the levels of enzymatic activity; while SBA and PNA were uneffective. We discuss these findings in relation to a possible role of glycosidases in controlling mesenchymal GAG turnover.     

Phospholipase C activity in cultured human skin fibroblasts

In this paper we report the detection of phospholipase C activity in cultured human skin fibroblasts by a rapid, sensitive method. Sonicates of fibroblasts were incubated with L-3-phosphatidyl-[U-14C]-inositol and the incubation mixture extracted with chloroform/methanol. The solvent components were then separated into 2 phases by the addition of 2 M KCl. Phospholipase C activity, determined from the amount of [14C] in the aqueous phase, agreed well with the enzyme activity assessed by other methods. The optimum pH for the enzyme was 7.0 and the enzyme was found to be dependent on Ca2+ and deoxycholate for optimal activity. The demonstration of phospholipase C activity by this method in cultured skin fibroblasts provides a useful means with which to study, in human tissues, the physiological control of this enzyme and its derangements in disease states in a controlled fashion.     

Age-related changes in collagenase expression in cultured embryonic and fetal human skin fibroblasts

Since skin collagenase is required for initiation of the degradation of types I and III collagens, the major collagens of the human dermis, we examined its expression during embryonic and fetal development. When using skin fibroblasts cultured from human embryos and fetuses, immunoreactive collagenase concentrations were strongly correlated with estimated gestational age (p less than 0.001), with levels at 7-8 weeks of gestation that were about one-twentieth of those in the 29-week cell cultures. In crude culture medium, the apparent catalytic efficiency (activity per unit immunoreactive protein) was variable, an observation attributable in part to variable expression of a collagenase-inhibitory protein. Following chromatographic purification, four of ten fetal collagenases were found to have greater than or equal to 4-fold decrease in specific activity, suggesting that these particular fetal collagenases may be structurally and/or catalytically altered. Since the decreased levels of immunoreactive protein suggested that decreased enzyme synthesis was the major mechanism, we examined collagenase synthesis in a cell-free translation system. Here, we quantitated collagenase expression in the culture medium of intact cells prior to harvesting mRNA. Compared with the intact adult cells, the fetal cells had 3-17 times less collagenase activity in the medium, while in cell-free translation there was a 2- to 3-fold decrease in collagenase synthesis. These data suggest that decreased in vitro expression is correlated with decreased levels of translatable collagenase mRNA but that other factors, such as the collagenase inhibitor and altered specific activity of the enzyme, may be important in modulating collagenase activity.     

Alpha-L-iduronidase activity in cultured skin fibroblasts and amniotic fluid cells

Using phenyl-α-l-iduronide as substrate, we have examined the level of α-l-iduronidase activity in homogenates of fibroblasts derived from normal individuals, from patients affected with α-l-iduronidase deficiency disorders (Hurler syndrome, Scheie syndrome, and a disease of intermediate severity presumed to be a Hurler/ Scheie compound) and from parents of such patients. Extracts derived from the affected individuals had no detectable α-l-iduronidase activity, whereas those derived from heterozygotes varied between 20% and 95% of the normal mean. Overlap between normal and heterozygous levels was reduced if the α-l-iduronidase activity was expressed on the basis of the β-galactosidase activity in the same homogenate. Cultured amniotic fluid cells from normal pregnancies had less than half as much α-l-iduronidase activity as fibroblasts from normal adults; this might cause problems in distinguishing a heterozygous fetus from an affected one by the enzyme assay alone.     

Developmental changes in glycosaminoglycans,collagen and collagenase activity in embryonic chick skin

In order to study remodelling of connective tissue during development, changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin at various stages have been studied.Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20).At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis.Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

Somatomedin-like bioactivities of a growth hormone fragment on embryonic chick cartilage and cultured human fibroblasts

Somatomedins are growth hormone-dependent peptides which appear to mediate many of the effects of growth hormone in vivo. These peptides are commonly assayed by their enhancement of proteoglycan sulfation in cartilage (or fibroblasts). We now report that fragment A-II (bovine growth hormone, 96–133) stimulates sulfation in chick embryonic cartilage and cultured fibroblasts. Enhancement of fibroblast proteoglycan sulfation by fragment A-II was log-dose dependent with maximal stimulation at 10^?8 M. The 25–100% maximal enhancement by fragment A-II was similar to that reported with a preparation of somatomedin A (Wasteson, A., Uthne, K., Westermark, B. (1973) Biochem. J. 136, 1069–1074). Sulfation of chick cartilage, in the presence of both serum (hypopituitary human) and fragment A-II was greater than the sum of the effects of each substance tested separately. Fragment A-II was tested between 10^?12 and 10^?8 M; maximal stimulation occurred at 5 · 10^?11 M. To our knowledge, no other growth hormone fragment has yet been shown to possess these somatomedin-like bioactivities. Our results suggest that fragment A-II may be very useful as a model peptide to study the actions and mechanism of naturally occurring sometomedins.     

Developmental changes in glycosaminoglycans, collagen and collagenase activity in embryonic chick skin.

In order to study remodeling of connective tissue during development, changes in glycosaminoglycan, collagen and collagenase activity in embryonic chick skin at various stages have been studied. Collagen content in the skin increased rapidly during days 14 to 18, then leveled off until hatching. Prior to the increase of collagen deposition in the skin, a sharp decrease in chondroitin sulfate was observed between days 11 and 14, while dermatan sulfate increased almost 4 fold during days 12 to 14, then increased steadily until hatching. Hyaluronic acid decreased progressively during the stages investigated (days 11 to 20). At the same stage as the rate of collagen deposition in the tissue became maximal (day 16), the amount of dialyzable hydroxyproline showed a maximum, indicating that an increased rate of collagen deposition in the tissue was accompanied by accelerated collagenolysis. Culture of skin from various stages of embryonic development revealed that 16 day old tissue was potentially capable of secreting the highest levels of collagenase. This collagenase was mostly inactive against soluble collagen and collagen fibrils but could be activated by 3 M NaSCN treatment.     

Protein turnover in senescent cultured chick embryo fibroblasts

The over-all rates of protein synthesis, degradation and net accumulation were estimated in rapidly growing young and slowly doubling old cultures of chick fibroblasts. We find that not only the rate of protein synthesis is reduced in senescent cultures, but the average rate of protein degradation is also slowed down considerably. This decrease in the rate of protein breakdown in aging cells stands in contrast with the previously observed acceleration of this process by other conditions (such as serum deprivation or overcrowding) that lead to the cessation of cellular growth. Though the retarded protein degradation may contribute to the acculation of abnormal proteins in senescent cells we find that the breakdown of grossly abnormal puromycin peptides proceeds equally rapidly in young and old cultures. The protein content of senescent cells increases by 1.8-fold as compared to young cells, while the average cell volume is increased even more (almost 5-fold). By contrast, consideration of the over-all balance of protein metabolism in these cells indicates that the average concentration of metabolically turning-over proteins is somewhat higher in senescent than in young fibroblasts.     

Exogenous glycosaminoglycans modulate extracellular and intracellular accumulation of newly synthesized glycosaminoglycans by embryonic chick skin fibroblasts

The effects of exogenous glycosaminoglycans (GAG) on newly synthesized GAG accumulation in intra- and extra-cellular compartments of 17 incubation days chick embryonic skin fibroblasts have been examined. Exogenous GAG are able to act on both total GAG synthesis and secretion by embryonic fibroblasts and on the relative concentration of individual GAG. A decline in hyaluronic acid and an increase in chondroitin sulfate plus dermatan sulfate in the cellular compartment for all GAG administered have been detected. There was also similar behaviour in the extracellular compartment after sulfated GAG administration.     

Effects of phenytoin on acetylcholinesterase activity and cell protein in cultured chick embryonic skeletal muscle

Cultured pectoral muscle from 11-day-old chick embryos was treated for 48 h with phenytoin (diphenylhydantoin, DPH) in concentrations ranging from 15 to 270 microgram/ml on days 7-9 in vitro. Acetylcholinesterase (AChE, EC 3.1.1.7), creatine phosphokinase (CPK, EC 2.7.3.2), and lactic dehydrogenase (LDH, EC 1.1.1.27) activities, [3H]leucine incorporation into protein, and total protein of the cultures decreased in a dose-related manner with DPH concentrations of 30 microgram/ml and greater. Total AChE activity and AChE activity released into the medium were specifically decreased with 15 microgram DPH per millilitre. In cultures treated chronically with 15 microgram DPH per millilitre on days 5-13 in vitro, total AChE activity and AChE activity released into the medium were 66.0 +/- 13.2 and 64.7 +/- 11.8% of untreated controls, respectively, but cellular AChE activity, cell protein, and [3H]leucine incorporation into protein were unaffected. The results indicate that DPH specifically decreases the total net synthesis of AChE activity by a direct action on cultured chick embryo muscle.     

Intracellular activity and secretion of various lysosomal glycosidases in cultured human skin fibroblasts

The intracellular activities of four lysosomal glycosidases (alpha-L-fucosidase, beta-D-hexosaminidase, beta-D-galactosidase and beta-D-glucuronidase) in human skin fibroblasts cultured in a medium with 0.1% serum increased in a greater degree than that in a medium with 10% serum. Only two glycosidases (alpha-L-fucosidase and beta-D-hexosaminidase) were secreted by fibroblasts in the culture medium. The extracellular activity of alpha-L-fucosidase and beta-D-hexosaminidase was equivalent to 80 and 25% of their intracellular activity in serum-sufficient fibroblasts and 40 and 15%--in serum-restricted fibroblasts. These results suggest that the observer phenomena are controlled by the levels of autophagy, endocytosis and membrane recycling.     

beta-Glucoside hydrolase activity of normal and glucosylceramidotic cultured human skin fibroblasts.

Cultured human skin fibroblasts from normal and glucosylceramidotic subjects are found to contain one beta-glucoside hydrolase as compared with multiple enzymes in other tissues. The fibroblast enzyme has an approximate molecular weight of 150,000 under isotonic conditions, as determined by gel filtration. It occurs as a large aggregate at low ionic strength. Ceramide, 4-methylumbelliferyl, and p-nitrophenyl beta-glucosides are active as substrates. The enzyme in whole cell homogenates is membrane-bound and is solubilized by a combination of Triton X-100 and sodium taurocholate. It has a pH optimum at 4.2 and no demonstrable divalent cation requirement. The cultured fibroblast beta-glucosidase displays close similarity to one of the forms of beta-glucosidase in human spleen, specifically that form which is affected in Gaucher's disease. 4-Methylumbelliferyl beta-glucosidase activity in homozygous fibroblasts from infantile and adult forms of Gaucher's disease are reduced to 9 and 14%, respectively, of normal fibroblast activity. The residual activity in the lipidotic cells shows increased heat lability, but cannot be distinguished from that in normal cells with respect to gel exclusion properties, Michaelis constant, and pH dependence.     

Effects of electrically induced contractile activity on cultured embryonic chick breast muscle cells

Development of chicken breast muscle is characterized by the sequential appearance of six electrophoretically distinct myosin heavy chain (HC) isoforms. Cultured secondary myotubes, derived from 12-day embryonic chick breast muscle, mainly express the early embryonic HC isoform HCemb/e, normally present in 8-day embryonic breast muscle, and the two fast light chain isoforms LC1f and LC2f. Direct low-frequency (2.5 Hz) stimulation of these myotubes via platinum electrodes leads to a shift in myosin HC expression with increases in the late embryonic HC isoform HCemb/l amounting to 35% of total HC in 19-day-stimulated cultures. Measurements of 35S-methionine incorporation and immunohistochemical analyses demonstrate increases in LC3f. This increase is also seen at the mRNA level. These results indicate that induced contractile activity promotes myotube maturation in vitro. The observation that chronic stimulation enhances the expression of the slow isoform LC2s at the RNA, as well as the protein level, suggests an additional effect consisting of a fast-to-slow change in phenotype expression. In view of the fact that muscle maturation and phenotype expression is under neural control during development in vivo, our results on directly stimulated, aneural myotubes indicate that neurally transmitted contractile activity may be an important factor in modulating phenotype expression of secondary myotubes.     

Self-contact inhibition of movement in cultured chick heart fibroblasts

Collisions between two lamellar processes extended from a single locomoting cultured cell were examined by time-lapse cinemicrography and transmission electron microscopy. In most cases after contact the forward movement of either one or both of the lamellae ceased and was followed by a phase of retraction of the lamellae resulting in the breaking of the contact. The events correspond well to the contact inhibition of movement expressed when two cells collide. The similarity is also shown in the ultrastructure of the cell contacts which exhibit a close parallel arrangement of the apposed cell membranes and an alignment of microfilaments in the regions of the cytoplasm at the contacts.