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PubMed1992

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.

Animals↗
PubMed1989

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.

Animals↗
PubMed1983

Inaccessibility of certain Ricinus lectin binding sites due to the increase in hyaluronic acid during chick embryo development.

Chick embryo fibroblasts constitute a useful model for investigating cell surface differentiation using Ricinus lectin as a marker. Fibroblasts from 8-day chick embryos had two classes of Ricinus lectin binding sites, whereas those from 16-day embryos displayed only one class. Hyaluronidase treatment of fibroblasts from 8-day embryos had no effect on their capacity to bind Ricinus lectin; however after this treatment, 16-day cells resembled 8-day cells since the former also exhibited two classes of lectin-binding sites. Treatment with hyaluronidase released 2-5 times more hyaluronic acid from the older cells than from the younger cells. The same hyaluronidase treatment did not change the number of 8-day cells detached by trypsin from the substrate, but increased the number of detached 16-day cells. These observations suggest (i) that the greater adhesiveness to the substrate of the 16-day cells might be due to the presence on the cell surface of a larger amount of glycosaminoglycans at 16 days than at 8 days, and (ii) that the increased accumulation of hyaluronic acid on the cell surface might be involved in an alteration in the cell membrane during differentiation.

Animals↗
PubMed1992

Experimental studies on cardiovascular disorders and cellular Ca handling in shell-less chick embryos.

1. Chick embryos rendered Ca deficient by long-term culture outside the eggshell develop hypertension/tachycardia. 2. In this study, the hypertension of the shell-less (SL) chick embryo has been characterized by comparing their cardiovascular response to adrenergic drugs and cellular Ca handling function with normal (NL) embryos. 3. Before and after the administration of noradrenaline (NA), phentolamine (PA), isoproterenol (IP), and propranolol (PP), blood pressure and pulse rate were measured. 4. Baseline blood pressure was consistently higher in SL embryos. In both embryos, blood pressure was elevated by NA and PP, but lowered by IP and PA. Pharmacological sensitivity to NA and PA was higher in SL embryos. Serum Ca was lower in SL embryos, while myocardial calcium content was not different. At the cellular level, Ca handling was studied using red blood cells (RBC) from SL and NL embryos under varying osmotic and ionic conditions by analysing 45Ca uptake and cell volume regulation. 5. Two features of RBC Ca uptake were apparent: (i) Na-Ca exchange was functional since Ca uptake was dependent on external Na; (ii) Ca uptake was dependent on total ionic electrochemical gradient. The SL RBC had greater ion permeability, more active Na-Ca exchange function and higher Ca pumping out capacity. 6. These systemic and cellular properties of the SL embryo indicate that it is a novel and useful experimental system to study the relationship between Ca homeostasis and the development of hypertension.

Adenosine Triphosphate↗
PubMed1977

Avascular and vascular phases of tumour growth in the chick embryo.

The chick embryo was used to study the relationship between the onset of tumour neovascularization and tumour growth. Walker 256 carcinosarcoma was implanted on the chrioallantoic membrane (CAM) of about 600 embryos aged 5-16 days. Tumour diameter and changes in the CAM vasculature in response to the implants were recorded daily. Representative tumours were examined by light microscopy of Epon-embedded tissue and autoradiography after injection of [3H]-thymidine. Tumours remained avascular for 72 h, after which they were penetrated by new blood vessels and began a phase of rapid growth. The rate of growth during this vascular phase was greatest for implants on 5- and 6-day-old embryos and decreased the later the day of implantation. The time of onset of tumour angiogenesis appears to be independent of the immunological state of the chick embryo, although the rate of growth after vascularization may be modified by the onset of immunity. This study suggests that the avascular and vascular phases of tumour growth are separable, and that the avascular tumour population lives under the growth constraints which limit the size of a tumour spheroid growing in soft agar or aqueous humour.

Animals↗
PubMed2002

Development of respiratory rhythms in perinatal chick embryos.

In chick embryos, gas exchange takes place via the chorioallantoic membrane (CAM) and the lungs at approximately 1 day prior to hatching. The present study was designed to elucidate the development of respiratory rhythms in the chick embryo during the whole pipping (perinatal) period with a condenser-microphone measuring system. The microphone was hermetically attached on the eggshell over the air cell on day 18 of incubation. It first detected a cardiogenic signal (i.e. acoustocardiogram), and then beak clapping and breathing signals (acoustorespirogram, ARG). The first signals of lung ventilation appeared intermittently and irregularly approximately once per 5 s among the clapping signals after the embryo penetrated its beak into the air cell (internal pipping, IP). The respiratory rhythm then developed irregularly, with a subsequent more regular rate. The envelope pattern of breathing from the onset of IP through external pipping (EP) to hatching was constructed by a specially devised procedure, which eliminated external and internal noises. The envelope patterns indicated that the IP, EP and whole perinatal periods of 10 embryos were 14.1+/-6.4 (S.D.), 13.6+/-4.0 and 27.6+/-5.4 h, respectively. In addition, they also indicated the period of embryonic hatching activity (i.e. climax) which was 48+/-19 min. The development of respiratory rhythm was also shown by the instantaneous respiratory rate (IRR) which was designated as an inverse value of two adjacent ARG waves.

Animals↗
PubMed1995

Development of rhythmic melatonin synthesis in cultured pineal glands and pineal cells isolated from chick embryo.

The chick pineal gland exhibits circadian rhythms in melatonin synthesis under in vivo and in vitro conditions. A daily rhythm of melatonin production was first detectable in pineal glands isolated from chick embryos at embryonic day 16 and incubated under a LD cycle. All pineal glands isolated from 17-day-old and older embryos were rhythmic while no gland isolated at embryonic day 14 and 15 exhibited a daily rhythm in melatonin synthesis. Melatonin production in static cultures of embryonic pineal cells was rhythmic over 48 h if the cells were kept under a LD cycle. When embryonic pineal cells were incubated in constant darkness the rhythm in melatonin production was damped within 48 h. These results suggest that chick pineal cells from embryonic day 16 onwards are photosensitive but that the endogenous component of the melatonin rhythm is not completely developed at that age. A soluble analogue of cAMP stimulated and norepinephrine inhibited melatonin synthesis in cultured embryonic pineal cells. These findings indicate that the stimulatory and inhibitory pathways controlling melatonin synthesis in the mature pineal gland are effective in pineal cells isolated from chick embryos at least 2 days before hatching.

Age Factors↗
PubMed1985

Coordinate regulation of collagen and alkaline phosphatase levels in chick embryo chondrocytes.

Chick embryo tibial chondrocytes release into their extracellular matrix several species of proteochondroitin sulfate and collagen as well as matrix vesicles that are rich in Ca2+ and alkaline phosphatase and that appear to play a role in the calcification of cartilage. To determine whether there was any parallel regulation of the production of these products, the rates of collagen synthesis by cultured chick embryo tibial chondrocytes were altered, and the resulting changes in proteochondroitin sulfate synthesis and alkaline phosphatase levels in the cells were measured. As the rate of collagen synthesis was increased by adding increasing amounts of ascorbic acid to the culture medium, there was a parallel increase in the level of alkaline phosphatase. Similarly, when the rate of collagen synthesis was inhibited by adding 3,4-dehydroproline to the culture medium, the levels of alkaline phosphatase fell. The alkaline phosphatase in the culture medium was associated with vesicles which appeared to be matrix vesicles. It was recovered quantitatively by filtration through membranes with a pore size of 0.1 mu and measured by solubilizing the alkaline phosphatase from the membrane with detergent and assaying with 4-methylumbelliferyl phosphate as the substrate. When the matrix vesicles from the culture medium were analyzed for collagen types, it was found that only Type X collagen was recovered in this fraction. The implications of the association of Type X collagen and the matrix vesicles, both of which are found primarily in growth plate cartilage in the zone of hypertrophied chondrocytes which is in the process of mineralization, are discussed.

Alkaline Phosphatase↗
PubMed2003

Effects of 1,2-dibromoethane on haematopoiesis in the chick embryo.

1. Chick embryo in ovo was used to investigate the effects of 1,2-dibromoethane (DBE) on haematopoiesis at a developmental stage where the primitive erythroid cells divide and differentiate in circulation. 2. Early after DBE treatment on embryonic day 3, annexin V/propidium iodide labelling showed acute cell death of erythroid elements, which was subsequently compensated for by the release of immature cells into the circulation. Simultaneously, the comet assay indicated increased DNA damage in DBE-exposed blood cells when compared with controls. 3. After embryonic day 5, there was no indication for ongoing prominent cell death in the DBE-treated group. However, the DNA damage assessed by the comet assay persisted until embryonic day 10 in the peripheral blood cells, and for even longer in cells from thymus and bursa. 4. The kinetics of DNA fragmentation in both erythroid and lymphoid cells implied genotoxic damage by DBE to the stem cells of the definitive elements and transmission of this damage through the successive cell generations. 5. The early chick embryo provides a suitable alternative to mammalian models for investigation of long-term effects of xenobiotics on haematopoiesis.

Animals↗
PubMed1996

Chick embryos as an alternative experimental animal for cardiovascular investigations: stable recording of electrocardiogram of chick embryos in ovo on the 16th day of incubation.

Recording of electrocardiogram (ECG) tracings in developing chick embryos often fails because of spontaneous motion of the embryos in the egg shell. We attempted to record ECG of chick embryos in ovo. When we injected a mixture of 450 mg/ml urethane and 45 mg/ml alpha-chloralose into the air sac of fertile eggs at volumes of 0.1 to 0.3 ml, the spontaneous motor activity of chick embryos was decreased and stable ECG tracings could be obtained from at least 10 min after the injection. The P, QRS, and T waves were noted in the electrograms, and the QT interval was positively correlated to the RR interval. The heart rate (HR) could be analyzed for the RR interval in fertile eggs after the 8th day of incubation. The HR of the 16-day fertile embryos was linearly increased with incubation temperature in the range from 31 to 41 degrees C. Using this system, cardiac effects of some drugs were examined. Isoprenaline and acetylcholine increased and decreased the HR in a dose-dependent manner, respectively, and these effects were inhibited by respective antagonists, propranolol and atropine. These ECG responses of chick embryos were similar to those of mammals or humans. In conclusion, stable ECG tracings could be obtained from chick embryos anesthetized by urethane and alpha-chloralose in ovo and this method may be applicable for the investigation of the developing heart and the evaluation of cardiovascular drugs.

Age Factors↗
PubMed1984

On auxotrophy for pyrimidines of respiration-deficient chick embryo cells.

Chick embryo cells treated with chloramphenicol are inherently resistant to the growth-inhibitory effect of the drug when cultured in the presence of tryptose phosphate broth. The cells were found to be auxotrophic for pyrimidines and the presence in the broth of compounds of pyrimidine origin is demonstrated by chromatographic procedures and mass spectral analyses. They are in the form of ribonucleosides, ribonucleotides and pyrimidine-containing oligoribonucleotides. To understand the mechanism responsible for pyrimidine auxotrophy, the activity of enzymes involved in the pyrimidine biosynthetic pathway was determined. Measurement of the conversion of dihydroorotic acid to orotic acid in cell-free extracts revealed that chloramphenicol-treated chick embryo cells are deficient in dihydroorotate dehydrogenase activity. The data in vitro are supported by studies on the nutritional requirements of the respiration-deficient cells and by the incorporation in vivo of labelled dihydroorotic acid into the acid-insoluble fraction of the cells. Although the activity of the dehydrogenase in vitro is decreased by 95%, the enzyme is present in chloramphenicol-treated cells and its activity is unmasked by the artificial electron acceptor menadione. A study of the activity of other enzymes of the pyrimidine biosynthetic pathway demonstrated that their activity is comparable to that in control cells. The present results indicate that auxotrophy for pyrimidines results from the inhibition of the flow of electrons along the mitochondrial electron transport chain.

Animals↗
PubMed1979

Production of erythropoietic colony-forming units and erythrocytes during chick embryo development: an attempt at modelization of chick embryo erythropoiesis.

The enumeration of erythropoietic colony-forming cells in vitro has allowed us to complete previous data on changes in the various erythroid cell populations during chick embryo-genesis. Erythrocytic colony-forming units in culture (CFU-cE) which are sensitive to avian erythropoietin appear in the blastoderm as soon as the 24th hour of development. They represent most likely precursors of the megalocytic erythropoiesis, and do not seem to derive from stem cells common with normocytic erythropoiesis. Data concerning vitelline normocytic erythropoiesis were analysed in a kinetic model based on stochastic change of the stem cells. From this model it appears that 17-20 cell divisions are required for differentiation of erythrocytes from stem cells.

Animals↗
PubMed1980

[Effect of some electric signals transmitted by an induction coil on weight increase, incorporation of marker, and histological and ultrastructural appearance of the skeleton in a chick embryo].

Embryo Chicks were exposed to various types of electrical impulses transmitted by induction coils between the 6th and 13th day of incubation. Compared with controls, the Chick tibias showed a highly significant increase in weight and length as well as increased incorporation of tritiated thymidine. In addition, the total weight of the treated embryos was significantly greater than that of the controls. The results suggest that the effects of these electro-magnetic fields, particularly their skeletal effects, act principally on cellular multiplication and/or enhanced cellular activity following an increase in protein synthesis.

Animals↗
PubMed1963

RIBOSOMAL RNA IN THE DEVELOPING CHICK EMBRYO.

Developing chick embryos from the stage of primitive-streak formation make ribosomal (28S and 16S) RNA. The size and composition of the RNA appears to be constant throughout the first 7 days of development.

Animals↗
PubMed1977

[ATP and ATPase activity in the liver and brain cell fractions of gamma-irradiated chick embryos and newly hatched chicks].

Studied were the level of ADP and the adenosinetriphosphatase in nuclei, mitochondria, and microsomes taken from the brain and liver of singly gamma-irradiated at 1000 rd chick embryos and birds. As a result of the treatment of ADP content dropped, while the activity ADP rose. These changes were most strongly expressed in the nuclei, then in the mitochondria, and to a lesser extent-in the microsomes. Twelve-day chick embryos showed more markedly expressed radiosensitivity than newly hatched chicks. This embryonal state is characterized by intense growth, differentiation and metabolic processes in the liver, which substantiate not only the higher radiosensitivity of this age group but the more strongly expressed changes in the liver as compared with the brain.

Adenosine Triphosphatases↗
PubMed1994

Expression of the galactose-binding lectins during the formation of organ primordia in the chick embryo.

Early chick embryos contain two beta-galactoside-binding lectins of 16 kDa and 14 kDa. using several antisera to these proteins, we have studied lectin expression at embryonic stages when the segregation and early differentiation of organ primordia are taking place. With antisera to the 16 kDa lectin that display similar immunoreactivity in immunoblot analysis, we show that these antisera exhibit varying immunoreactivity in embryo sections. One antiserum reacts preferentially with a matrix form of lectin while another detects mainly a cellular form of this protein. During early development, galactoside-binding lectins of the matrix type are expressed in the vitelline membrane, the outer and inner limiting membranes of the neural tube, the surface of the notochord and the coelomic surface of the cardiac rudiments. The cellular form of the lectin occurs in the intracellular yolk of early embryos, in the primordial germ cells, the myocardium, in the early myotome, and in a cohort of cells which are presumed to belong to the neural crest. Our results indicate that, although all of the antisera recognize the intracellular lectin of the extraembryonic endoderm, some antisera to the 16 kDa lectin exhibit preferential reactivity with different lectin isoforms. The extracellular matrix form of lectin is transiently expressed during early development at the stages when the segregation of organ primordia is occurring. It's expression could be related to the acquisition of polarity in developing epithelia. Results also suggest that various versions of the same protein may perform distinct developmental roles in the embryo.

Animals↗