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In vitro evaluation of plasticizer activity on the growth and metabolism of chick embryo fibroblasts and on the development of chick embryo lungs.

Administration of di(2-ethylhexyl) phthalate (DEHP) to primary cultures of chick embryo fibroblasts brought about a decrease in cell proliferation rate after 48 h and an inhibition of both DNA and protein synthesis measured by [3H]thymidine and [3H]leucine, respectively, after 48 h. The growth of chick embryo lung rudiments in vitro was also depressed by DEHP treatment. Lung rudiment were smaller in DEHP-treated embryos after 6 days' treatment. These results indicate that DEHP has a cytostatic effect on embryonic cells and tissues.

Animals

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

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

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

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

[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

[Induced degeneration and lysosome activity in the cells of the tail bud in the early chick embryo].

In chick embryos, cells of the tail bud are induced to degenerate by autophagocytosis when treated by puromycine or sodium fluoride. Although these degenerative processes exhibit some differences, according to the nature of the inhibitor used, their initial steps, which consist in a modification of the Golgi zone, are identical. These facts are discussed in relation to the problem of morphogenetic cell degeneration.

Animals

Morphological and biochemical differentiation in RSV transformed chick embryo myoblasts.

Chick embryo myoblasts have been transformed with a temperature sensitive mutant of Rous Sarcoma virus (RSV ts68). At permissive temperature (36 degrees C) it is shown that transformed myoblasts have lost their ability to form myotubes as well as to express the biochemical markers of myogenic differentiation. Upon a shift to the non-permissive temperature (41 degrees C), the normal program of differentiation is restored; myotubes are formed which express muscle specific proteins.

Acetylcholine

Cellular RNA and influenza-virion RNA are synthesized from different pyrimidine-nucleoside-triphosphate pools in chick-embryo cells.

Chick embryo cells infected with an influenza A (fowl plague) virus have been labelled with (3H)-uridine for different lengths of time. Virion RNA and cellular RNA have been separated by specific hybridization with a surplus of unlabelled viral complementary RNA and RNase digestion. The ratio of the specific radioacticity in the UMP and CMP moieties of both types of RNA has been determined. Since the rate of approach to equilibrium of CMP to UMP labelling of both types of RNA is completely different it is concluded that cellular and virion RNA are synthesized using different pyrimidine nucleoside triphosphate pools.

Animals

Glucose depletion accounts for the induction of two transformation-sensitive membrane proteinsin Rous sarcoma virus-transformed chick embryo fibroblasts.

Chick embryo fibroblasts transformed by Rous sarcoma virus have an increased content of two membrane proteins of molecular weights 78,000 and 95,000. The increased content of the 95,000-dalton protein and the principal increase in the content of the 78,000-dalton protein are not an early consequence of cell transformation but instead are secondary to the rapid depletion of glucose from the growth medium of transformed cells. When glucose is maintained at high levels in the growth medium of transformed cells, the synthesis of the 95,000-dalton protein is arrested and that of the 78,000-dalton protein is markedly suppressed. Upon removal of glucose from the growth medium of normal cells, these proteins increase to levels comparable to those of transformed cells. Because the amount of these two proteins is influenced by the presence or absence of glucose, we suggest they be referred to as "glucose-regulated proteins." GRP-78 and GRP-95. These proteins may have an important role in regulating the utilization of glucose in cultured cells.

Animals

Histogenesis of experimental open neural defects in the early chick embryo.

The chick embryo is a useful experimental model for investigating neural dysraphism. Windowing at 26 h incubation is by itself teratogenic, resulting in predominantly neural tube defects. A histological study of a regular series of specimens between pre-neurulation and later stages was undertaken. Open brain defects occurred at every stage after the expected closure of the anterior neuropore, suggesting that they arose by non-closure, myeloschisis was preceded by a characteristic triangular shape of the rhomboid sinus. Serial sections revealed regular open defects, with separation between the neural plate and tail-bud sources of neural tissue, but continuity of the neural plate into the caudal region. These findings suggest that myeloschisis arises by nonclosure of the neural folds. The establishment of myeloschisis was followed by local separation of the notochord from an open area of neural tube, but not by overgrowth of neural tissue. Myelodysplasia appeared at about the time of expected closure of the rhomboid sinus. Serial sections revealed irregular open defects, with complete absence of neural plate material and formation of the cord tissue from tailbud material alone. The lesions were accompanied by extensive cystic and hemorrhagic changes in local mesoderm, with reduction in somite volume. There was no associated notochordal separation.

Animals

Acute inflammatory response to cowpox virus infection of the chorioallantoic membrane of the chick embryo.

The chick embryo chorioallantoic membrane was used to study the acute inflammatory response in the absence of contributions from the immune system. In preliminary experiments, lesions of wild-type cowpox virus strain Brighton (CPV-BR) and a 38K gene deletion mutant of CPV-BR (CPV-BR.D1) were compared with vaccinia virus (strains WR and Copenhagen), fowlpox virus, laryngotracheitis virus, and infectious tenosynovitis virus, and were ranked for degree of induced inflammation. The maximal and minimal inflammatory responses were observed with CPV-BR.D1 and CPV-BR viruses, respectively. CPV-BR.D1 lacks a 38K gene which encodes an anti-inflammatory 38-kDa protein that has homology to SERPINs. The kinetics and character of the inflammatory response were examined further in the wild-type CPV-BR and mutant CPV-BR.D1 infections using cell counts, electron microscopy, and assays for inflammatory cell activation. CPV-BR virus infection rapidly spread through the ectoderm, uniformly infecting all cells with the production of large amounts of virions and viral-induced cytopathic effect, but evoking little or no inflammatory response until 144 hr p.i. The CPV-BR.D1 infection, on the other hand, was rapidly contained by a dexamethasone-sensitive inflammatory response mainly of activated heterophils which was advanced by 36 hr p.i. Both infections resulted in disseminated disease with similar numbers of liver lesions and only a slight difference in the LD50, with the CPV-BR.D1 values being higher than that for CPV-BR virus. In this model, the acute inflammatory response alone is unable to prevent disseminated disease and associated mortality.

Acute Disease

Antibody-mediated immuno-regulation in chick embryos and baby chicks.

Two- and three-week old chicks that received at hatching serum from either unimmunized birds or from chickens immunized with bovine serum albumin responded with normal hemagglutinin titers to immunization with mouse erythrocytes (MRBC). Other chicks administered at hatching or as embryos with homologous serum high in anti-MRBC activity responded poorly to MRBC immunization. When donor serum samples collected on different days after immunization were tested, the immunosuppressive capacity was negligible the first two days, increased in potency thereafter to a maximum level by the end of the week. The increase with time in the inhibiting property of the donor serum paralleled the serum anti-MRBC hemagglutinin response profile. The immune capacity of the antibody-treated recipients was severely depressed for over two weeks. By the fourth week, however, the immune responsiveness to MRBC, as measured by serum hemagglutinin titers and production of antigen sensitive units, had recovered to near normal levels.

Animals