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.     

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.     

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.     

Immunofluorescent visualization of 100 A filaments in different cultured chick embryo cell types.

Antibody prepared against the 55,000 dalton subunit of reconstituted chick gizzard 100 A filaments (anti-G55K) bound to the 100 A filaments of chick smooth muscle, cardiac muscle, and skeletal muscle cells, and to the 100 A filaments of Schwann cells and satellite glial cells of the peripheral nervous system. Anti-G55K did not bind to replicating presumptive myoblasts, fibroblasts, chondroblasts, pigment cells, neurons, or to central nervous system glial cells. This contrasted with the wider range of binding of antibody to the 58,000 dalton subunit of chick fibroblast 100 A filaments (anti-F58K) which bound to the 100 A filaments of all cell types examined except hepatocytes and skin epithelial cells. Anti-G55K) staining revealed a morphologically distinct distribution of 100 A filaments in the three types of muscle cells. Spindle shaped smooth muscle cells exhibited dense fluorescent staining near the poles of the cells, and also exhibited unique patches of fluorescent material after cytochalasin B and Colcemid treatment. In myotubes, the fluorescence was limited to longitudinal bundles of filaments between the striated myofibrils. Cardiac cells contained uniformly distributed fine filaments. Lastly, smooth muscle cells in various phases of mitosis bound the anti-G55K, whereas replicating presumptive skeletal myoblasts failed to bind the anti-G55K.     

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.     

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.     

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.     

The effects of dihydropyridine calcium antagonists on heme biosynthesis in chick embryo liver cell culture

A variety of 1,4-dihydropyridine calcium antagonists were tested for porphyrinogenic activity in chick embryo liver cell culture. 3,5-Dimethoxycarbonyl-1,4-dihydro-2, 6-dimethyl-4-(ortho-nitrophenyl)pyridine (nifedipine) was shown to be a potent porphyrinogenic agent. This activity was shared by a number of related analogues, viz., the 4-phenyl, 4-(meta-nitrophenyl), 4-(para-nitrophenyl), 4-(ortho-methoxyphenyl), 4-(meta-trifluoromethylphenyl), and 4-(para-trifluoromethylphenyl) analogues and nitrendipine; nicardipine exhibited very weak activity. The porphyrinogenic potency of the 1,4-dihydropyridines did not parallel their calcium antagonist activity. Nifedipine did not exhibit ferrochelatase-lowering activity in chick embryo liver cell culture and uroporphyrin and heptacarboxylic acid porphyrin were the major porphyrins to accumulate. Nifedipine did not cause suicidal destruction of cytochrome P-450 in chick embryo hepatic microsomes. Because nifedipine possesses comparable porphyrinogenic activity to sodium secobarbital in chick embryo liver cell culture, caution is required if nifedipine or related drugs are administered to patients with hereditary hepatic porphyria.     

Kinetics of carrot somatic embryo development in suspension culture

Somatic embryos in liquid culture can serve as a mass cloning system in a plant propagation program. A quantitative formulation of embryo development obtained from cell suspension cultures is used to develop a segregated kinetic model. The model is based on standard classification schemes as previously developed by plant physiologists. Dependent variables include carbohydrate concentrations (sucrose, fructose, and glucose) and biomass apportioned among the inoculum (free single cells, cell clusters), normal developmental stages, and aberrant cell and embryo types. Good agreement between the model and experimental results is indicated and allows for a rigorous approach to media optimization and reactor scaleup for embryo formation.     

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.