Proteins specified by Sindbis virus in chick embryo fibroblast cells

Large amounts of high molecular weight polypeptides were detected in “aged” chick embryo fibroblast cells infected with Sindbis virus. These polypeptides were shown to be virus specific by several criteria. At least some of the above polypeptides were shown to be precursors to smaller viral structural proteins by a pulse-chase experiment.     

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.     

Reversible regulation by magnesium of chick embryo fibroblast proliferation

The rate of 3H-thymidine incorporation and of cell proliferation in chick embryo fibroblast cultures are reduced coordinately when the [Mg2+] of the external medium is reduced below the physiological concentration of about 0.8 mM. These effects of moderately reduced [Mg2+] and the accompanying change in appearance of the cells, resemble the effects produced by lowering the [serum] of the medium. Cells subjected to severe Mg2+ deprivation, especially at low [Ca2+], die and detach from the culture dish. Cells kept at a reduced rate of proliferation for three days by moderate Mg2+ deprivation are quickly restored to rapid proliferation upon restoration of the normal [Mg2+] of the medium. The rate of proliferation of the chick embryo cells is reduced markedly by lowering [Ca2+] about 100-fold, but unlike the case of Mg2+-deprivation this can occur without significant effect on the rate of 3H-thymidine incorporation. More severe Ca2+ deprivation, which does lower the rate of 3H-thymidine incorporation, produces retraction of cells from one another and from the dish, and results in a distinctly abnormal, rounded appearance. The results lend weight to the thesis that free [Mg2+] plays a central role within the cell in the coordinate control of metabolism and growth. They also suggest that the effects produced by varying [Ca2+] in the medium are caused by changes at the external surface of the cell.     

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.     

The neural tube origin of ventral root sheath cells in the chick embryo

The embryonic origin of peripheral nerve Schwann/sheath cells is still uncertain. Although the neural crest is known to be an important source, it is not clear whether the ventral neural tube also contributes a progenitor population for motor axons. We have used the techniques of immunohistochemistry, electron microscopy and quail-chick grafting to examine this problem. Immunohistochemistry with monoclonal antibody HNK-1 identified a cluster of immunoreactive cells in the sclerotome, at the site of the future ventral root. With the electron microscope, nucleated cells could not be seen breaching the basal lamina of the neural tube, exclusively in the region of the ventral root and preceding axon outgrowth. After grafting a length of crest-ablated quail neural tube in place of host chick neural tube, a population of quail cells was found localized to the ventral root exit zone, associated with the ventral root axons. Taken together, these observations support the possibility of a neural tube origin for ventral root sheath cells, although we found no evidence for a more extensive migration of these cells. The ventral root cells share certain phenotypic traits, such as HNK-1 immunoreactivity, with neural-crest-derived Schwann cells, but are not necessarily identical to them. We argue that while they may help motor axons to exit the neural tube at the correct position, they are unlikely to guide axons beyond the immediate vicinity of the neural tube.     

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.     

The chick embryo fibroblast cytosolic DNA complex--a possible cell-cell messenger

1. The uptake of [3H]thymidine and [3H]uridine labelled DNA-RNA cytosol complex has been studied in chick embryo fibroblast cells. 2. The complex appears to be cleaved into DNA and RNA containing fragments in the recipient cell nucleus: both then enter the cell cytosol fraction, but the RNA fragment in particular is rapidly degraded. 3. Although [3H]thymidine labelled material present in the nucleus co-extracts with bulk nuclear DNA, caesium gradienting shows little or no evidence that integration of host and imported DNA has occurred. 4. It is suggested that the cytosolic/extruded DNA complex may be a "messenger" DNA, capable of the transfer of regulatory information between cells on a transient basis.     

Hyaluronan-dependent pericellular coats of chick embryo limb mesodermal cells: induction by basic fibroblast growth factor

Basic fibroblast growth factor (FGF) has been shown previously to be present in the chick embryonic limb during early stages of its development, at which time the limb mesodermal cells are proliferating within a hyaluronan-rich extracellular matrix. In this study, basic FGF was found to stimulate hyaluronan synthesis and production of hyaluronan-dependent pericellular coats by mesodermal cells from the chick embryo limb; acidic FGF, platelet-derived growth factor, epidermal growth factor, and retinoic acid either had a much smaller effect than basic FGF or an inhibitory effect. Transforming growth factor-beta stimulated hyaluronan synthesis and coat formation but, unlike basic FGF, this factor also stimulated chondroitin sulfate production by the mesodermal cells.     

The characteristics of the stimulation of the proliferation of cultured chick embryo cells during reseeding

The growth rate of chick embryo cells in slowly proliferating cultures is activated after substitution of conditioned medium by fresh one with serum. After cell replanting, the stimulation of cell proliferation takes place in both media with the same effectiveness. In serum-less medium replanted cells do not adhere to glass and die. The results suggest that cells reversibly lose their dependence upon serum growth factors and population density, as a result of replanting, but retain anchorage-dependence for growth.     

Chromomycin A3 inhibits influenza a virus multiplication in chick embryo fibroblast cells

The multiplication of Ulster 73 virus, an avian strain of type A influenza virus, was blocked in chick embryo fibroblast cells, CEF, by treatment with 0.5 microg/ml of chromomycin A3 whereas in LLC-MK2 cells no inhibition of replication was observed. Virus-induced polypeptide synthesis in chick embryo fibroblast cells was confined to the synthesis of PB2, PB1 and PA subunits of the RNA dependent-RNA polymerase, the nucleoprotein NP, the non-structural protein NS1, the haemagglutinin HA, the non-structural protein NS2; only the membrane M1 polypeptide synthesis was greatly inhibited. Viral unpolyadenylated cRNAs synthesis was studied at a late time of the infection, 8 hours p.i.: chromomycin A3 was able to inhibit the "novo" synthesis of complementary RNA poly(A)- and segment 7 of virion RNA. The mode of action of the drug in chick embryo fibroblast cells is discussed.     

The regulation of the intracellular sodium ion concentration in cultured chick embryo heart cells.

Using digital imaging microscopy with the fluorescent indicator sodium-binding benzofuran isophtalate, we examined the cytosolic Na+ concentration ([Na+]i) in individual chick embryo heart cells. Inhibition of the Na(+)-H+ exchanger using Na(+)-free (Li+ substituted) medium and inhibition of the Na(+)-efflux through the Na(+)-Ca2+ exchanger using Ca(2+)-free medium didn't change the [Na+]i. The opening of voltage-dependent Na+ channels with veratridine (150 micrograms/ml) and inhibition of the Na(+)-K(+)-Cl(-)-cotransporter with bumetanide (10 microM) led to an increase in [Na+]i by 107% and 86%, respectively, suggesting that the Na+ channels and the Na(+)-K(+)-Cl- cotransporter predominantly regulate the [Na+]i in cultured chick embryo heart cells.     

The search for the DNA of the chick embryo fibroblast cytosolic complex

1. Conventional DNA extraction procedures have failed to release free DNA from the chick embryo fibroblast cytosolic DNA-RNA complexes. 2. Free DNA has been released only from the smallest cell cytosol DNA fraction, which is not in the native state associated with RNA: it is very small (of the order of 100 bases) and single stranded. 3. However, phenol extraction does separate complex DNA-associated material from the RNA which has invariably been found to accompany it in all but the smallest fraction (see 2 above). 4. The principal factor preventing DNA release appears to be a massive aggregation of partially purified DNA-associated material.     

Epiblast origin and early migration of neural crest cells in the chick embryo

Chick embryos carrying transplants labeled with tritiated thymidine demonstrate that the neural crest originates in the anterior epiblast, at the junction of areas destined for epidermis and neural tube. As the neural tube begins to fold and the axis lengthens, cells along this junction are drawn dorsomedially; at the seven-somite stage they begin to separate from the epithelium of the head, and migrate into the angle between the epidermis and the neural tube. The paraxial mesoderm already populating this angle originates in more posterior and medial portions of the epiblast than do the neural crest cells; after invagination at the primitive streak, it migrates anterolaterally, ventral to the ectoderm layer, until it too is folded dorsomedially into the angle between the epidermis and the neural tube.     

Proteoglycan biosynthesis by chick embryo retina glial-like cells

In this report we present biochemical evidence that purified cultures of chick embryo retina glial-like cells actively synthesize heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) proteoglycans as well as hyaluronic acid. Glial-like cell cultures were metabolically labeled with [3H]glucosamine and 35SO4, and the medium, cell layer, and substratum-bound fractions were analyzed separately. Proteoglycans were characterized according to charge, apparent molecular size, and glycosaminoglycan (GAG) composition and were found to be differentially distributed among the cellular compartments. HS was the predominant GAG overall and was the major species found in the cell layer and substratum-bound fractions. CS/DS was also present in each fraction and comprised the largest proportion of GAGs in the medium. The major GAG-containing material resolved into three different size classes. The first, found in the cell layer and substratum-bound fractions, contained both CS/DS and HS and was of large size. A second, intermediately sized class with a higher CS/DS:HS ratio was found in the medium. The smallest class was found in the cell layer fraction and comprised HS, most likely present as free GAG chains. In addition, each fraction contained hyaluronic acid. Characteristics of these macromolecules differ from those produced by purified cultures of chick embryo retina neurons and photoreceptors in terms of size, compartmental distribution, and presence of hyaluronic acid.     

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.     

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.     

Concanavalin A-binding by cells of the early chick embryo

The surfaces of cells from the early embryo of the chick were examined using electron microscope techniques for the visualization of concanavalin A-binding sites. Horseradish peroxidase and Ferritin labelled concanavalin A were used to determine the distribution of the binding sites. All surfaces of the epiblast and hypoblast layers which were accessible to concanavalin A showed the presence of binding sites in stage 1 embryos. The ventral surface of the epiblast showed a high lectin affinity which may reflect the development of a basal lamina on this surface. The individual hypoblast cells at this stage showed a non-uniform distribution of binding sites, having a greater affinity on the dorsal surface than the ventral. By the time of primitive streak formation (stage 4-5) the dorsal surface of the epiblast displayed increased binding sites, while the frequency of sites on the ventral surface of the endoblast was reduced. The latter may reflect a change from one cell population to another, which occurs in the lower layer of the embryo at this time. No consistent correlation could be drawn between changes in motility of cells actually invaginating through the primitive streak and changes in affinity for concanavalin A. An overall increase in affinity of the dorsal surface of the epiblast was revealed by Ferritin and may reflect the changes in surface structure occurring in readiness for the morphogenetic migrations of gastrulation.     

The origin and distribution of cartilage canals in the chick embryo.]

113 femurs and 111 tibias originating from chick embryos 8-21 days after incubation were injected with Indian ink and made transparent. The origin and distribution of cartilage canals are described. The existence of a pattern of segmental distribution, atypical canals and anastomosis between epiphysial canals and diaphysial marrow processes is reported.