Antiviral factor production by infected chick embryo fibroblasts]

The secretion of antiviral factor (AF) by infected cell cultures was examined. Activity of AF depended on the cell culture used. AF produced by infected chick embryo fibroblasts had maximal activity. No activity was registered in BHK-21 cells, whereas human embryo fibroblasts and cell line Vero produced a low level of activity. Actinomycin D and cycloheximide prevented the production of AF. The results indicate that VEE virus-infected chick embryo fibroblasts produce AF which may be attributed to nonspecific factors of cell defense.     

Changes in phospholipids from chick fibroblasts during embryo development

The content of cholesterol and total phospholipids was assayed in 8- and 16- day old chick embryo fibroblasts, harvested at subconfluence after a 48- and 96-hour primoculture, respectively. Cholesterol content did not change during embryo development, whereas the amount of total phospholipids decreased (28%) from the 8th to the 16th day of development, giving an increase of the cholesterol/phospholipid ratio. Studies of the fatty acid composition of the predominant membrane phospholipids indicated that there was no significant change in phosphatidylcholine, whereas phosphatidylethanolamine was depleted in the myristate, as the embryo grew older. These findings demonstrate that the lipid contents are modified during embryo development and suggest that the fluidity of chick embryo cell membranes decreased during development.     

Sequestered actin in chick embryo fibroblasts

Chick embryo fibroblasts contain about 75-100 M unpolymerized actin and at least four proteins which can bind actin monomers, actin depolymerizing factor (ADF), gelsolin, profilin, and thymosin ₄ (T₄). Fibroblast extracts are analyzed by non-denaturing polyacrylamide gel electrophoresis and immunoblotting where most of the G-actin is detected as a complex with T₄. When fibroblast extracts are fractionated by gel filtration and the fractions are analyzed by PAGE and HPLC, most of the G-actin elutes in a peak that also contains T₄ at an overall molar ratio of 1.9:1 relative to actin. Gelsolin, profilin, and ADF are also detectable in the gel filtration eluate and at least partly coelute with actin, and account for only a minor fraction of the soluble actin pool. These observations indicate that under the growth conditions studied, T₄ is the major actin-sequestering protein in fibroblasts.     

Growth inhibitory activity in extracts from human fibroblasts

Indirect evidence for the presence of a growth inhibitor in normal human fibroblasts has been obtained previously; the inhibitory activity has been found associated with crude cell extracts, but the molecule responsible for the growth inhibition has never been isolated. We have isolated a glycopeptide fraction from human fibroblast cultures, whose synthesis decreases when the cells are stimulated into the division cycle. It was separated by electric charge, lectin affinity, and molecular mass. When added to quiescent cells simultaneously with a growth stimulus, the glycopeptide reduces DNA synthesis activity. The relationship of the kinetics of the synthesis of the glycopeptide with the cell division cycle and its molecular weight are different from what has been described so far for other growth regulators. The decreased synthesis of this inhibitor, induced by growth factors, seems to be one of the requirements for the initiation of the division cycle by human fibroblasts. This response to growth factors was stable during the lifespan of the fibroblast population and became less pronounced only in cells at the end of their replicative potential. © 1993 Wiley-Liss, Inc.     

Properties and purification of a glucose-regulated protein from chick embryo fibroblasts

A glucose-regulated protein of molecular weight 78 000 (GRP-78) had been purified from a membrane fraction isolated from viral transformed chick embryo fibroblasts. Purification was achieved by extraction of the membrane fraction with Triton X-100, and chromatography on diethylaminoethyl-cellulose and hydroxyapatite. The purified protein exhibited one single spot on two-dimensional polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has a pI of about 5.3. A monospecific antiserum to GRP-78 was generated in a goat. Immunofluorescence studies using affinity purified antibodies to GRP-78 revealed that this protein was not exposed on the cell surface but was localized in a granular vesicular network inside the cell that resembles the distribution of endoplasmic reticulum. The availability of purified GRP-78 and a specific antiserum to it should prove useful in elucidating the role of this protein in glucose metabolism and its relationship to malignant transformation.     

Phospholipid turnover in hamster and chick embryo fibroblasts

The stability of the glycerol backbone of phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl serine was measured in growing and non-growing hamster and chick embryo fibroblasts. Major differences were found for the rates of degradation of the individual glycerophospholipids in both hamster and chick embryo fibroblasts: considerable degradation of phosphatidyl choline was detected over a 24 h period while at the same time no degradation of the glycerol backbone of phosphatidyl ethanolamine and phosphatidyl serine was observed. The patterns of stability of these glycerophospholipids were similar in growing and non-growing cells.     

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.     

Substructure of membranes in cultivated chick embryo fibroblasts

Summary Electron microscope examination of the plasma membrane of chick embryo fibroblasts cultured in vitro revealed the presence of a single osmiophilic layer about 90 Å thick and a substructure composed of ovoid sub-units associated with an amorphous component. These ovoid sub-units measured approximately 112 Å along the major axis and 75 Å along the minor axis and were composed of a central core, approximately 30 Å by 60 Å, surrounded by a peripheral component.Examination of other membranous components of these cells revealed a similar ovoid subunit structure in a single layered membrane. Differences in thickness and in the sizes of ovoid sub-units were seen in these membranes. The ergastoplasmic membranes, the outer nuclear membranes, the outer mitochondrial and the Golgi membranes were found to be the thinnest.These varied in thickness from approximately 75 Å to 80 Å. The thickest membranes seen were the inner nuclear membranes. These were approximately 100 Å thick. The dimensions of the ovoid sub-units corresponded with differences in the thickness of the various membranes. These findings support the concept of a particulate substructure of cell membranes.This work was aided by Research Grant PH 5593 from the National Science Foundation. Some of the equipment used was purchased with funds from the National Institutes of Health Grant 2TI GM 326. I wish to thank Dr. Robert M. Dougherty from the Department of Microbiology who grew and supplied me with the chick embryo fibroblast cultures used in these studies, and Mrs. Ursula Feller fer her technical assistance.     

Properties and purification of a glucose-regulated protein from chick embryo fibroblasts.

A glucose-regulated protein of molecular weight 78,000 (GRP-78) had been purified from a membrane fraction isolated from viral transformed chick embryo fibroblasts. Purification was achieved by extraction of the membrane fraction with Triton X-100, and chromatography on diethylaminoethyl-cellulose and hydroxyapatite. The purified protein exhibited one single spot on two-dimensional polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has a pI of about 5.3. A monospecific antiserum to GRP-78 was generated in a goat. Immunofluorescence studies using affinity purified antibodies to GRP-78 revealed that this protein was not exposed on the cell surface but was localized in a granular vesicular network inside the cell that resembles the distribution of endoplasmic reticulum. The availability of purified GRP-78 and a specific antiserum to it should prove useful in elucidating the role of this protein in glucose metabolism and its relationship to malignant transformation.     

Growth-inhibitory glycopeptides obtained from the cell surface of cultured chick embryo fibroblasts

Cell surface glycopeptides were obtained from cultured chick embryo fibroblasts (CEF) by digestion with Pronase E, and a fraction exerting growth-inhibitory activity on CEF was isolated by high performance gel permeation chromatography. The active fraction, tentatively termed cell surface glycopeptide-2 (CSGP-2), was soluble in 5% trichloroacetic acid (TCA) or 75% ethanol. It inhibited the growth of CEF reversibly at 10-20 micrograms sugar/ml, but did not inhibit BALB/c mouse 3T3, SV40-transformed 3T3, and human diploid cells at similar concentration. The growth-inhibitory activity of CSGP-2 was reduced or lost after digestion with neuraminidase or oxidation with sodium metaperiodate. Cellulose acetate electrophoresis revealed that CSGP-2 was a mixture of sialoglycopeptides. A similar growth inhibitor was also isolated from chicken embryonic tissues.     

Counter-transport in chick embryo fibroblasts. A significant factor in measurement of glucose entry.

Enhanced rates of carrier-mediated 3-O-methyl-D-glucose (0.1 mM) transport were observed in primary cell cultures of chicken embryo fibroblasts deprived of glucose for 1 day. The addition of 5.5 mM-glucose, glucosamine or 2-deoxy-D-glucose for 15 min (37 degrees C) to glucose-starved cultures followed by washing and immediate measurement of 3-O-methyl-D-glucose transport resulted in an apparent further stimulation of transport. Transport stimulation increased with increasing concentrations of the added preincubation sugar and was observed at test concentrations ranging from 0.1 mM- to 10 mM-3-O-methyl-D-glucose. This enhancement occurred when the preloaded sugar was rapidly effluxing from cells and was eliminated by allowing cultures to incubate in buffer without sugar for 30 min (37 degrees C) after the removal of hexose and before measuring transport. A transient overshoot in the cumulative uptake of 3-O-methyl-D-glucose was observed in glucose-starved cultures that were pre-incubated in the presence of 55 mM-glucose or -glucosamine for 15 min (37 degrees C). These data suggest that counter-transport accounts for the apparent enhancement of glucose-transport capability observed in glucose-starved cells when they are briefly re-exposed to hexose.     

Collagen formation by fibroblasts of the chick embryo dermis

This investigation has sought to determine the relation between collagen fiber and fibroblast during fibrogenesis. Toward this end the surfaces of chick fibroblasts grown under in vitro conditions have been examined with the electron microscope after fixation in OsO(4). Supplementary information has been obtained from thin sections of fibroblasts fixed in situ during phases of fiber production. The evidence provided by these studies and by various conditions of the experiments indicates that the unit fibrils of collagen form in close association with the cell surface. They were never observed within the cell. When these unit fibrils form in bundles it appears as though templates of some nature, possibly coinciding with stress fibers within the cell cortex, influence the polymerization of the fibrils out of material available at the cell surface. From here the fibrils and bundles of them are shed into the intercellular spaces and there grow to limited diameters by accretion of materials from the general milieu.