On the adaptation of cultured chick embryo cells to growth in the presence of chloramphenicol

We have found that tryptose phosphate broth (TPB) prevents the inhibitory effect of chloramphenicol (CAM) on the cell proliferation of chick embryo fibroblasts. Study of growth parameters indicated that no lag or adaptation period appeared necessary for TPB-exposed chick cell populations to grow in the presence of CAM suggesting that a particular cell type was not selected. TPB did not prevent the inhibitory effect of CAM on the mitochondrial protein-synthesizing system. This was supported by cytochrome oxidase activity measurements, studies on the incorporation of 35S-metionine into mitochondrial proteins, electron microscopic observation of alterations in mitochondrial structure. Oxygen consumption was reduced by 95% and cyanide, 2-4-dinitrophenol, and salicylhydroxamic acid do not significantly affect the residual respiration. Analyses of reduced-minus-oxidized-cytochrome spectra of CAM-treated chick cells demonstrate the disappearance of the absorption bands of cytochromes aa3, b559, c1, and c. The presence of a type b cytochrome with maxima at 552 and 557 nm was observed. The results obtained indicate that long-term cultures of CAM-treated chick embryo cells cultivated in the presence of TPB grow with mitochondria devoid of a functional respiratory chain.     

The effect of calcium and inhibitors on corn mitochondrial respiration

The effects of KCN, antimycin A, malonate, rotenone, and amytal on the oxidation of malate, succinate, and extramitochondrial reduced nicotinamide adenosine dinucleotide (NADH) by corn mitochondria were studied. Potassium cyanide and antimycin A inhibited the oxidation of all three substrates. Rotenone and amytal inhibited only the oxidation of malate, and malonate inhibited only the oxidation of succinate. Rotenone, amytal, and malonate did not inhibit the oxidation of extramitochondrial NADH. The calcium stimulation of the oxidation of extramitochondrial NADH was prevented by KCN and antimycin A but not by amytal, rotenone, or malonate. It is suggested that corn mitochondria possess a flavoprotein specific for extramitochondrial NADH and that this flavoprotein is sensitive to divalent cations.     

Contact stimulation of the proliferation of cultured chick embryo cells

It was reported elsewhere (Gasparian, Grigorian, 1989a) that in the secondary chick embryo cell culture, after its superinoculation with additional homologous cells, the cell population density increased and the cell proliferation was activated. It has been shown in the present paper that the cell proliferation rate increase after inoculation of fixed cells, as well. The results obtained are discussed as a direct evidence for growth-stimulating effect of contacts between homologous cells.     

Differential effect of hypertonic initiation block on the synthesis of collagen chains by cultured chick embryo cells

The major type of collagen synthesized by fibroblasts or bone cells, type I collagen, consists of two chains normally found in a 2:1 ratio designated alpha 1(I)2 alpha 2(I) or more simply alpha 1(I)2 alpha 2. I have analyzed the relative synthesis of type I chains in these cells under conditions which reduce the initiation of protein synthesis. It was found that in bone cells, which make a large amount of collagen, the alpha 1(I):alpha 2 ratio is unaltered whereas in fibroblasts, which make smaller amounts of collagen, the alpha 2 chain is particularly sensitive to these same conditions. Examination of the collagen secreted into the medium, under these same conditions, also revealed an altered chain ratio from cells making low amounts of collagen.     

Study of some enzyme activities in cultured chick embryo brain nerve cells treated by chick embryo brain extracts

Brain extracts from 8-day-old chick embryos have been shown to influence morphological development of dissociated brain cells from 7-day-old chick embryos in culture. Stimulatory, effects on size of the neuronal somas and on growth of long processes were observed by adding the cytosol of the brain extract or the dialysate of the cytosol. These morphological changes parallel modifications of various enzyme activities according to the age of the cultures. Adenyl cyclase, (Na⁺, K⁺)- and Mg²⁺-ATPase, 5-nucleotidase, choline acetyltransferase, and acetylcholinesterase activities were studied between 5 and 14 days of culture. Adenyl cyclase activity was strongly stimulated at 8 days by both extracts. (Na⁺, K⁺)-and Mg²⁺-ATPase activities were stimulated in 8-day-old cultures only by the dialysate. 5-Nucleotidase activity was stimulated in 8-day-old cultures by the dialysate and in 11-day-old cultures by both extracts. Choline acetyltransferase activity was stimulated by the cytosol in 8-day-old cultures and by the dialysate in 11-day-old cultures. The total acetylcholinesterase activity was higher in 8-, 11-, and 14-day-old cultures treated with the cytosol. When the cells were treated with the dialysate, the activity was only higher in 14-day-old cultures. We also found that following the addition of brain extracts, the specific activity of the enzymes we studied was enhanced and became close to the values found in vivo during embryogenesis. Thus in parallel to the morphological modifications observed in nerve cell cultures treated by embryo brain extracts, biochemical variations especially involved in synaptogenesis and membrane development could be measured.     

3,4-Dihydroxyphenylalanine in cultured ventricular cells from chick embryo heart

Abstract— —A substance resembling catecholamine found in chick hearts during the early stages of embryologic development was identified as DOPA. Cell cultures and fluorescence microscopy indicated intracellular location of this substance in myocardial cells. The absence of nerve tissue in the cell cultures was demonstrated by electron microscopy.     

Adenosine transport by cultured glial cells from chick embryo brain

The transport of adenosine was studied in pure cultures of glial cells from chick embryo brain. In order to avoid complications in uptake measurements due to adenosine metabolism, cultures were depleted of ATP by incubation with cyanide and iodoacetate prior to addition of [³H]adenosine. Under the 5- to 25-s periods used for the transport assay, no adenosine metabolism could be detected. Initial rates of adenosine transport under these conditions obeyed the Michaelis-Menten relationship with K_m = 370 μM and V_max = 10.3 nmol/min/mg cell protein. ATP depletion or elimination of Na⁺ from the assay medium had no significant effect on initial rates of adenosine uptake. However, when assays were carried out under conditions of significant adenosine metabolism (10-min uptake in the absence of metabolic inhibitors), a high-affinity incorporation process could be demonstrated in the glial cells (K_m = 12 μM; V_max = 0.34 nmol/ min/mg protein). The transport activity expressed in ATP-depleted glial cells was most sensitive to inhibition by nitrobenzylthioinosine, dipyridamole, and N⁶-benzyladenosine. In decreasing order of potency, N⁶-methyladenosine, 2-chloroadenosine, inosine, and thymidine also blocked adenosine translocation in glial cultures. Thus, adenosine transport by cultured glial cells occurs by means of a low-affinity, facilitated diffusion system which is similar to the nucleoside transporter in cells of nonneural origin.     

Further studies on the extrusion of cytosol macromolecules by cultured chick embryo fibroblast cells

The RNA and lipid-associated macromolecules extruded by chick embryo fibroblast cells have been analysed by chromatography. The results taken with those previously obtained suggest that cultured chick embryo fibroblasts excrete a wide range of cytosol macromolecules into the surrounding medium. As previously found with DNA and protein, the chromatographic patterns found with supernatant and cell cytosol preparations were closely similar, suggesting that the media supernatant macromolecules are cytosal derived. A proportion of the RNA and lipid associated material elutes at the same position as the previously observed for DNA and protein on gel exclusion chromatography, suggesting that cell cytosol contains a discrete DNA-RNA-protein lipid complex (size approx 5 X 10(5) dalton) which is extruded by cultured cells.     

Tryptose phosphate broth confers to chick embryo cells resistance to the inhibitory effect of chloramphenicol on growth

Tryptose phosphate broth (10% V/V) prevents the inhibitory effect of chloramphenicol (80 ug/ml) on the growth of chick embryo fibroblasts. Cellular cytochrome $\text{c}$ oxidase activity is rapidly lost indicating that chloramphenicol inhibits the mitochondrial protein-synthesizing system in the presence of the broth. To compensate for the apparent decreased mitochondrial ATP production, pyruvate kinase and lactate dehydrogenase activities, 2-deoxyglucose uptake and lactic acid produced in treated cells are greater than those in the control cells during the first few generations. These fermentation values tend to revert to normal during later cell generations.     

Influence of various extracellular matrix components on the behavior of cultured chick embryo dermal cells

Dermal cells isolated from the back of 7-day chick embryos were cultured on homogeneous two-dimensional substrates consisting of one or two extracellular matrix components (type I, III or IV collagen, fibronectin and several glycosaminoglycans: hyaluronate, chondroitin-4, chondroitin-6, dermatan or heparan sulfate). The effect of these substrates on cell behavior was compared with that of culture dish polystyrene. Three parameters of cell behavior were examined: cell proliferation and patterning, spreading (cell surface) and locomotion (velocity and directionality). Data were collected by sequential microphotography and analyzed by computer assisted morphometry. Types I and III collagen, hyaluronate and heparan sulfate had a slowing down effect on cell proliferation and patterning. The inhibitory effect of type I collagen was also detected in mixtures with glycosaminoglycans. The other components had no effect. While the smallest spreading was observed on fibronectin substrate, the largest was recorded on chondroitin-6 sulfate and heparan sulfate. The slowest velocity of locomotion was measured on fibronectin, types I and IV collagen and a mixture of type I collagen and chondroitin-6 sulfate. The fastest speed was recorded on chondroitin-4 sulfate. These effects are discussed in view of our knowledge of the role of the dermis in the development of skin and cutaneous appendages, and in the light of the morphogenetically related microheterogeneous distribution of collagens, fibronectin and various glycosaminoglycans in the developing skin.     

Studies on the cytosolic DNA of chick embryo fibroblasts and its uptake by recipient cultured cells

Cytosol and extruded DNA complexes from cultured chick embryo fibroblast cells have been separated by agarose gel chromatography at intervals after pulse labelling with [3H]thymidine. The proportion of the various cytosol components changed markedly with time: there was a lag period of 3 hr before the major labelled (5 X 10(5) dalton) DNA complex appeared in the cytosol, and a further lag period of 5 hr before it was extruded from the cell. Cultured chick embryo fibroblast, and rat spleen, cells rapidly and very efficiently import their own or each others cytosolic DNA complexes into their respective cytosol fractions: the material recovered from the cytosol of recipient cells is characteristic of the presented material. Homologous cytosolic DNA complex presented to chick embryo fibroblast cells also becomes associated with the nucleus. The rat at which this occurs is comparable with the rate of incorporation of [3H]thymidine into nuclear DNA.     

The cytosol origin of macromolecules extruded by cultured chick embryo fibroblast cells

The DNA and protein extruded by chick embryo fibroblast cells has been analysed by chromatography. A high proportion of the DNA is in the form of a protein-complex of size around 5 X 10(5) dalton. The patterns of the DNA and protein extruded into the supernatant are closely similar in many respects to those found in the cell cytosol. It is concluded that the macromolecular material extruded by cells in culture is of cytosol origin: a possible function in terms of "information" carriage is proposed.