Functional and structural characteristics of polyribosomes associated with chick embryo cell nuclei

Polyribosomes bound to the outer nuclear membrane was isolated from purified preparations of chicken embryo cell nuclei. These polyribosomes were shown to consist fractions forming unstable complexes with the nuclear membrane which can be separated from the latter by treatment with high ionic strength buffer solutions. Using sedimentation and gradient density analyses, the nuclei-bound RNP complexes were shown to be predominantly composed of 80S monosomes which take an active part in collagen polypeptide synthesis in cell-free protein-synthesizing systems. A comparison of sedimentation properties and collagen-synthesizing activity of nuclei-bound polyribosomes and cytoplasmic polyribosomes forming unstable complexes with endoplasmic membranes, it was concluded that the nuclei-bound 80S monosomes are an early step in the formation of cytoplasmic polyribosomes.     

Somatotropic cell differentiation in an organ culture of the chick embryo adenohypophysis

Morphogenetic potencies of the adenohypophysis tissue from 4.5 to 11-day old chicken embryos used for the differentiation of somatotropic cells were investigated by methods of organ tissue culture. STG-cells were detected in cultures by immunofluorescence using an antiserum to human STG. In vitro studies of organ cultures revealed differentiation of STG-cells when adenohypophysis tissue was cultured from the 5.5th, 7th, 9th and 11th day of development in the absence of the diencephalon. Differentiation of STG-cells occurred predominantly in embryo caudal lobe transplants after chorion-allantois culturing of Rathke's pocket fragments from 4.5-, 5.0- and 5.5-day old embryos. The data obtained suggest that at late stages of Rathke's pocket development differentiation of STG-cells is preprogrammed and that determined precursors of these cells are located in the caudal lobe of the germ.     

Myosin synthesis by fusion-arrested chick embryo myoblasts in cell culture.

The synthesis and accumulation of myosin was studied in subcultures of fusion-blocked, postmitotic embryonic chicken myogenic cells. Electron micrographs and fluorescent microscopy with antimyosin revealed that most, if not all, of these cells contain myosin. It was also found that these cells are capable of accumulating myosin at rates comparable to fused cells. Incipient T-tubule formation was also present in some of the blocked cells. It is concluded that cell fusion is not a prerequisite for myosin synthesis and accumulation or T-tubule formation during myogenesis in vitro.     

Kinetics of chick embryo cell types in culture

The growth kinetics and population doubling limits of chick embryonic fibroblasts, chondroblasts, and retinal pigment cells were compared. Chondroblasts were found to have a cumulative population doubling level (37 +/- 3 PDL) similar (p = 0.05) to that of control fibroblasts (42 +/- 2 PDL), in individual and pooled clones. While both cell types have similar doubling potential, the proportion of tritium-labeled nuclei decreases, and differs significantly as doubling level increases. This age-associated decline is due to an extension in the population doubling time. Direct cell-cycle analysis shows this increase to occur in the G1 phase. Furthermore, cartilage colonies maintain their phenotypic expression (metachromasia) throughout their lifespan under conditions of subcloning at sparse density. When fibroblasts derived from 15 day chick embryos are compared with fibroblasts from 10 day embryos (41 +/- 2 PDL) there is no significant difference (p = 0.05) in cumulative PDL or percent labeled nuclei, indicating that fibroblasts of different embryonic age have similar potential. The addition of hydrocortisone and insulin to the medium significantly shortens (25 +/- 2 PDL) the lifespan of 10 day chick fibroblasts. Kinetics of retinal pigment cells show a population doubling potential (29 +/- 1 PDL) different from fibroblasts and chondroblasts, suggesting that different cell types may not have similar limits on doubling potential when first determined in embryogenesis. Hydrocortisone and insulin have no effect on the growth kinetics or lifespan of retinal pigment cells in culture.     

The differentiation of prolactin cells in an organ culture of chick embryo adenohypophysis

We have studied differentiation of prolactin cells in explants of cephalic and caudal parts of Rathke's pouch of 4.5 day and 5.5 day old chick embryos after their incubation in vitro lasting for 7-8 days. Indirect immunofluorescence using an antiserum against bovine prolactin was used to detect prolactin cells in the cultures. Differentiation of prolactin cells was detected regularly in explants of the cephalic lobe of the adenohypophysis anlage in 5.5 day old embryos; under certain growth conditions prolactin cells were found in explants of the same lobe in 4.5 day old embryos. Prolactin cells were either absent or found in small numbers in cultures of the caudal part of adenohypophysis of 5.5 day old embryos. Our results provide evidence for the appearance of the committed precursors of prolactin cells in the Rathke's pouch at late stages of its formation and for their regional localization in the cephalic part of the anlage. This localization is in correspondence with the distribution of differentiated cells of this type in definitive adenohypophysis.     

Inhibition of chick embryo hepatic uroporphyrinogen decarboxylase by components of xenobiotic-treated chick embryo hepatocytes in culture. II.

A variety of xenobiotics, viz., 3,3',4,4'-tetrachlorobiphenyl (TCBP), sodium phenobarbital (PB), 3,5-diethoxycarbonyl-2, 4,6-trimethylpyridine (OX-DDC), and nifedipine, cause a decrease in uroporphyrinogen decarboxylase (UROG-D) activity, accompanied by uroporphyrin accumulation, in chick embryo hepatocytes in culture. In this study the activity of 17-day-old chick embryo hepatic UROG-D was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I, and it was shown that a UROG-D inhibitor, previously reported to accumulate in TCBP-treated and PB-treated chick embryo hepatocytes in culture, also accumulates in OX-DDC-treated and nifedipine-treated chick embryo hepatocytes in culture. It was concluded that the accumulation of a UROG-D inhibitor provides an explanation for the UROG-D inhibition observed in this culture system with xenobiotics that cause uroporphyrin accumulation. Studies of the UROG-D inhibitory fraction isolated from the 10,000 x g, 40,000 x g, and 100,000 x g supernatant fractions of cultured chick embryo hepatocyte homogenate led to the conclusion that the UROG-D inhibitor is derived from a soluble component of the homogenate.     

Enhanced growth and plaquing of rabies virus in static chick embryo cell culture.

The 7-day egg passage line of HEP Flury strain of rabies virus was inoculated to primary chick embyro (CE) cells prepared in different ways to compared efficiencies of viral growth and plaquing. Special care to minimize cellular damage due to trypsin at the step of monodispersion and sowing a comparatively large number of cells for monolayer preparation were required for rabies plaquing, whereas such cares were not necessary for plaquing of vesicular stomatitis virus. Plaque number and size were increased by incorporation of a high concentration of thymidine into cell growth medium. Various other means to produce a static state of CE cells were tested, and a maximal plaquing efficiency was obtained when dishes receiving a massive number of dispersed cells in MEM plus 1% calf serum were incubated at 37 C for 1 day without any buffering for monolayer preparation and postinfection incubation was done at 32 C in a CO2-incubator. Bottle cultures of CE cells prepared in a similar manner, when infected with HEP Flury virus, yielded a markedly higher titer of virus that CE cells prepared by our previous standard method.     

Correlation between the structural and functional differentiation of chick embryo cardiomyocytes in cellular aggregates

In order to establish the possible relation of the structural characteristics acquired by cardiomyocytes and their functional differentiation (spontaneous contractions), a study of the myofibrillogenesis on chick embryo at Hamburger-Hamilton stage 5 was carried out using techniques of dissociation and cellular reaggregation.     

Inhibition of chick embryo hepatic uroporphyrinogen decarboxylase by components of xenobiotic-treated chick embryo hepatocytes in culture

Uroporphyrinogen decarboxylase (UROG-D) activity in the 10,000g supernatant of 17-day-old chick embryo liver homogenates was determined by measuring the conversion of pentacarboxylporphyrinogen I to coproporphyrinogen I. The optimum pH of the enzyme was found to be approximately 6.0 and enzyme activity was found to be linear with protein concentrations ranging from 0.3 to 2.0 mg/mL. At a protein concentration of 1.2 mg/mL and pH 6.0, the activity was found to be linear for a reaction time of 50 min and to be approximately 10 pmol/(mg protein.min). This enzyme assay was used to demonstrate that a UROG-D inhibitor, previously reported to accumulate in rodent liver, also accumulates in 3,3'4,4'-tretrachlorobiphenyl (TCBP) and sodium phenobarbital (PB) treated chick embryo hepatocytes in culture. This results accords with the previous demonstration of a TCBP- and PB-induced decrease in UROG-D activity in this system. Uroporphyrin accumulation in chick embryo hepatocyte culture is interpreted as resulting from a combination of two mechanisms, viz., inhibition of UROG-D activity and uroporphyrinogen oxidation to uroporphyrin catalyzed by a cytochrome P-450 isozyme.     

Functional state of cultured chick embryo cell mitochondria investigated in situ

The cell membrane of cultured chick embryo cells is permeable to Ca²⁺ after treatment with mannitol. Ca²⁺ uptake by mannitol-treated cells can be attributed to the mitochondria. $\text{Ca}{}^{\mathrm{2+}}\text{O}$ quotients and acceptor control ratios of such cells and isolated mitochondria are identical.Digitonin exerts two time-dependent effects on mannitol-treated cells: it increases the maximum extent of Ca²⁺ uptake by 5 – 20 percent. It also partially uncouples respiration from Ca²⁺ accumulation.It is suggested that mannitol treatment of cultured cells provides an easy way to study Ca²⁺ uptake by mitochondria $\text{in situ}$.     

Spontaneous synaptic activity in cell culture of chick embryo spinal cord

The spontaneous development of synaptic activity (SSA) was studied in cell cultures of chick embryo spinal cord. The complicated time structure of the SSA, an important early-stage characteristic of which was giant inhibitory postsynaptic currents (IPSC), was demonstrated. The ionic nature and pharmacological sensitivity of these IPSC suggest that glycine is their transmitter. Emergence of excitatory postsynaptic currents (EPSC) and complex antagonistic relationships between excitatory and inhibitory SSA was detected later. Possible mechanisms for maintenance of synaptic activity during the inhibitory function are discussed. Correlations between the regularities of synaptic transmission development that we have disclosed and neuronal circuit electrical activity are examined.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 280–290, May–June, 1991.     

Induction of porphyrin synthesis in chick embryo liver cell culture by synthetic polychlorobiphenyl isomers

The effects of a series of synthetic di-tetra- and hexachlorobiphenyl isomers and commercial polychlorinated biphenyls on the porphyrin biosynthesis in chick embryo liver cells in culture were examined.It was found that 3,4,3′,4′-tetra- and 3,4,5,3′,4′,5′-hexachlorobiphenyl isomers were the most active inducers, which were approximately 20 times as active as 1,4-dihydro-3,5-dicarbethoxy-2,4,6-trimethylpyridine (DDC) in porphyrin production. 3,5,3′,5′-Tetra- and 2,3,4,2′,3′,4′-hexachlorobiphenyl isomers were moderate inducers, which were approximately 2.0 to 2.5 times as active as DDC. 2,4,6,2′,4′,6′-Hexachlorobiphenyl showed the same activity as DCC. Compounds such as 4,4′-di-, 2,3,2′,3′-, 2,4,2′,4′- and 2,6,2′,6′-tetrachlorobiphenyl were weak inducers and 2,5,2′,5′-tetrachloro- and decachlorobiphenyl isomers were found to be inactive. Kanechlor-400 was the strongest inducer among the commercial polychlorinated biphenyls investigated.The structural requirements for potent porphyrin-inducing activity of chlorobiphenyl isomers were found to be the para and meta substituted structure causing a more highly conjugated and nearly coplanar conformation. It was found that induction caused by some chlorobiphenyls was subject to feed-back repression by end-product heme. In addition, the metabolism of chlorobiphenyls in mice was influenced by the unsubstituted pairs of carbon atoms in the molecule. These results lead us to postulate the following hypothesis, namely, that strong inducers may displace heme directly and incorporate into a hydrophobic pocket of the apo-represor protein, thus causing an induction of δ-aminolevulinic acid synthetase.     

The effect of growth rate on differentiation of chick embryo limb bud mesenchyme in organ culture

Small explants of limb bud mesenchyme of day chick embryos which form muscle in organ culture synthesize proportionally less protein than DNA than do large explants which form cartilage. Chondrogenesis occurred in the central area of greatest population density in reaggregating limb bud cells, myotubes in areas of lesser density and fibroblasts in the sparsely populated periphery. Small explants grown in microdrops in plastic dishes undergo less cell division and form cartilage, but not muscle. Small explants on lens paper undergo more cell division and form muscle, but not cartilage.     

The expression of differentiation by chicken lens epithelium in in vitro cell culture

Dissociated cells of the lens epithelium of newly hatched chickens were cultured in vitro to investigate whether cells actively grown in culture retain their own differentive entiative traits to form lens fibers. After an exponential growth phase of the flattened epithelial cells, a number of “islets” of smaller epithelial cells with polygonal shape appeared. Along the periphery of these islets, the characteristic morphological change which leads to the formation of spherical bodies was observed. Electron microscopic observation showed the differentiation of lens fibers in these spherical bodies comparable to those in the lens in situ. Accumulation of δ-chrystallin was confirmed in such “lentoid” bodies. Outgrowth of the lens epithelial cells was maintained in in vitro culture up to about 50 days with several subculturings. The formation of lentoid bodies occurred in each subculture generation, which started from a homogeneous population of flattened epithelial cells. The present culture conditions permit the maintenance of such a population of cells that have a high growth potential and stably retains their differentiative trait to form lens fiber, even after repeated replication under in vitro conditions.