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Detection of Marek's disease virus serotype 1 (MDV1) glycoprotein D in MDV1-infected chick embryo fibroblasts.

Chick embryo fibroblasts (CEFs) infected with three strains of Marek's disease virus serotype 1 (MDV1), GA, Md5 and JM, were subjected to indirect immunofluorescence assay with monoclonal antibodies (MAbs) against MDV1 homolog of glycoprotein D (MDV1 gD) of herpes simplex virus. By the MAbs, a number of MDV1 gD-positive cells were detected in CEFs infected with GA, whereas only a few and no positive cells were detected in CEFs infected with Md5 and JM, respectively. The MDV1 gD in GA-infected CEFs was recognized as the band of 64 kDa in immunoblot analysis using one of the MAbs. This is the first report that the MDV1 gD was detected in MDV1-infected cell cultures.

Animals↗

Deprival of nicotinamide leads to enhanced glucose transport in chick embryo fibroblasts.

Chick embryo fibroblasts growing in medium free of pyridine ring precursors of NADH and NADPH replicate several times before multiplication ceases. The rate of glucose transport is progressively enhanced, finally reaching levels several times higher than those normally observed in cells severely depleted of NADH. Whereas normal cells respond to additional glucose by further reducing transport, the NADH-depleted cell is refractory to glucose even at five times the normal glucose concentration. Readdition of nicotinamide does little to restore normal transport within 24 h. On the other hand NAD+ or NADP+ provided simultaneously with glucose results in a sharp decline in measurable transport within 2-4 h. The role of the pyridine nucleotides in this reduction of transport function is for the moment unknown.

3-O-Methylglucose↗

Sequestered actin in chick embryo fibroblasts.

Chick embryo fibroblasts contain about 75-100 microM unpolymerized actin and at least four proteins which can bind actin monomers, actin depolymerizing factor (ADF), gelsolin, profilin, and thymosin beta4 (Tbeta4). Fibroblast extracts are analyzed by non-denaturing polyacrylamide gel electrophoresis and immunoblotting where most of the G-actin is detected as a complex with Tbeta4. When fibroblast extracts are fractionated by gel filtration and the fractions are analyzed by PAGE and HPLC, most of the G-actin elutes in a peak that also contains Tbeta4 at an overall molar ratio of 1.9:1 relative to actin. Gelsolin, profilin, and ADF are also detectable in the gel filtration eluate and at least partly coelute with actin, and account for only a minor fraction of the soluble actin pool. These observations indicate that under the growth conditions studied, Tbeta4 is the major actin-sequestering protein in fibroblasts.

Actins↗

[Ultrastructural aspects of vasculogenesis in the optic tectum of the chick embryo].

In chick embryos of 4,8,12 and 19 incubation days the ultrastructure of the intrinsic vessels of the optic lobes has been analyzed. During the early periods of the optic tectum vasculogenesis morphological differences have been observed between the vessels which, arising from the external vascular plexus, radially run through the mantle layer, and the vessels of the deep vascular plexus, which precociously arises by branching and anastomosing of the radial trunks immediately beneath the neuroepithelium. Such differences concerning the endothelium thickness, the characteristics of its luminal and abluminal outline, the time of appearance of the endothelial basement lamina and the glial layer, and the width of the perivascular spaces have been related to the growing patterns of the radial vessels which, at least during the first developmental days, are unbranched trunks and precociously differentiate elongating as the mantle layer become more and more thick, and, respectively, of the vessels of the deep plexus, the morphological maturation of which is belated owing to the continuous sprouting of new vascular sprouts.

Animals↗

Ontogeny of 5-aminolevulinic dehydratase and porphobilinogen deaminase activities in the yolk sac membrane and liver of chick embryos.

1. Chick embryos of 7, 9, 11, 12, 13, 14, 15, 17 and 19 d of embryonic development were examined to determine the activities of 5-aminolevulinic dehydratase (ALA-D, EC 4.2.1.24) and porphobilinogen deaminase (PBG-D, EC 4.3.1.8). 2. Liver and yolk sac membrane ALA-D specific activities showed a maximum between 12 and 13 d of embryonic development, yolk sac membrane PBG-ase activity a maximum at 9 d and at 7 d in liver. Total activities of ALA-D and PBG-D were not constant during the course of embryonic development but probably related to the changes of intensity of haem synthesis. 3. ALA-D and PBG-ase activities were higher in yolk sac membrane than in liver, showing the importance of the yolk sac membrane as erythropoietic tissue. PBG-D catalysed the rate-limiting reaction of the cytosolic steps in the biosynthetic pathway in both tissues.

Age Factors↗

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↗

A kinematic analysis of hindlimb motility in 9- and 10-day-old chick embryos.

Although chick embryonic leg movements appear jerky and disorganized, the underlying motor patterns are coordinated. This apparent conflict in results was investigated using kinematic analyses to provide detailed quantitative descriptions of leg movements during spontaneous motility in 9- and 10-day-old chick embryos. In many respects, hip, knee, and ankle movements were highly variable. There was variation in movement durations and in the number of leg joints that participated in a given movement. Motion could begin with either flexion or extension. Furthermore, the limb could return to its rest position between movements or move continuously to produce sequences of variable lengths. Patterns of interlimb coordination included alternating, synchronous and independent movements of the legs. We propose that these variable features account for the uncoordinated appearance of embryonic leg movements. In addition to variable features, however, some consistent characteristics were identified. For example, when more than one joint was active, activity typically began and ended synchronously. Furthermore, all active joints generally extended or flexed together and movements were symmetrical. These data are consistent with previous EMG results. Therefore, despite the variability in some parameters that results in the perception that embryonic leg movements are random and uncoordinated, our kinematic analyses show that an organized pattern of interjoint coordination is a prominent feature. This basic pattern shows some similarities to, but is less complex than, kinematic patterns found during a variety of posthatching behaviors.

Animals↗

Specific dephosphorylation of membrane proteins in Rous sarcoma virus-transformed chick embryo fibroblasts.

Chick embryo fibroblasts (CEF) infected with avian sarcoma virus become rapidly transformed as a result of expression of the viral src gene in the form of a single polypeptide of molecular weight 60,000 (pp60src) with protein kinase activity and suggested preferential association with the plasma membrane. Studies with normal avian and mammalian cells have revealed the presence of an antigenically related protein which seems to have similar kinase activity, but which is present at less than 1% of the levels of virally induced src protein found in transformed cells. As dynamic phosphorylation is important in numerous regulatory processes, the phenotypic expression of transformation may arise from an imbalance in one or more regulatory mechanisms that are controlled by protein phosphorylation. The cell membrane is affected during transformation, including its phosphotransferase activity. The latter has been shown using isolated membrane fractions whose properties may be changed during preparation. Therefore, we have compared the phosphorylation state of individual membrane proteins found in intact normal and RSV-transformed cells and report here the identification of two heavily phosphorylated, acidic membrane proteins in normal CEF which are specifically dephosphorylated on transformation by wild-type and temperature-sensitive Rous sarcoma viruses.

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↗

Biosynthesis of gangliosides in the developing chick embryo retina.

Chick embryo retinas were cultured with [3H]glucosamine on each day between days 6 and 12 of development. The total labeling of gangliosides decreased from day 6 to day 12. The decrease was mostly due to the decrease in the labeling of disialosyllactosylceramide (GD3), which diminished to less than 2%, while the labeling of the disialoganglioside GD1a decreased to about 30%. Labeling of endogenous gangliosides by incubating retina homogenates with CMP-[3H]N-acetylneuraminic acid, in addition to showing a decrease in the labeling of GD3, showed a twofold increase in the labeling of GD1a. The changes in the pattern of labeling correlated with the decrease of both the incorporation of [3H]thymidine into DNA and the activity of CMP-NeuAc:GM3 sialosyltransferase and with the increase of both the activities of UDP-Ga1NAc:GM3 N-acetylgalactosaminyltransferase and choline acetyltransferase. The results suggest that the shift in the pattern of labeling of gangliosides occurs in association with the transition from the proliferative to the nonproliferative state of neuronal cells. Retinas at each age cultured with [3H]glucosamine for 22 hr maintain the radioactivity in gangliosides for at least 4 additional days in culture. This indicates that within this developmental interval there is not a period characterized by having a high turnover rate of gangliosides.

Age Factors↗

A moderate decrease in temperature inhibits the calcium signaling mechanism(s) of the regulatory volume decrease in chick embryo cardiomyocytes.

Chick cardiomyocytes, when submitted to hyposmotic swelling, exhibit a partial regulatory volume decrease (RVD). A Ca2+ influx by stretch-activated channels signals a taurine efflux and the RVD at 37 degrees C. We evaluated the cell's performance at room temperature. Cardiomyocytes isolated and cultured from 11-day-old chick embryos were submitted to a hyposmotic solution (180 mOsm/kg H2O) at 37 degrees C and at room temperature (26 degrees C). Under these conditions we measured the changes in cell volume as well as the intracellular free Ca2+ (using fura-2). During hyposmotic swelling, cells at 37 degrees C displayed a peak relative volume of 1.61 +/- 0.03 and recovery to 1.22 +/- 0.04 (N = 14), while cells at 26 degrees C presented a peak swell relative volume of 1.74 +/- 0.06 and did not recover (1.59 +/- 0.09, N = 9). Transient increases in intracellular Ca2+, which are characteristic of the normal RVD, were observed at both temperatures (29.1 +/- 4.5% (N = 8) and 115.2 +/- 42.8% (N = 5) increase at 37 degrees and 26 degrees C (P<0.05), respectively). A delay in the Ca2+ transient increase was also observed when the cells were at 26 degrees C (109 +/- 34 s compared to 38 +/- 9 s at 37 degrees C, P<0.05). At room temperature the RVD does not occur because the calcium transient increase, which is an early event in the signaling of the RVD, is delayed. Also, free calcium is not cleared as in the 37 degrees C RVD. In the normal RVD the free calcium returns to baseline levels. The very high and persistent free calcium levels seen at room temperature can lead to unregulated enzyme activities and may promote irreversible injury and cell death.

Animals↗

Studies on the induction and phosphorylation of xanthine dehydrogenase in cultured chick embryo hepatocytes.

Chick embryo hepatocytes, cultured in a chemically defined medium, were used to investigate hormonal requirements for xanthine-dehydrogenase induction and to determine whether the enzyme is phosphorylated. Triiodothyronine is found to be required to induce the synthesis of active enzyme. Inclusion of sodium tungstate in the medium resulted in the complete loss of enzyme activity but no decrease of immunochemically detectable levels of enzyme. Immunoprecipitated xanthine dehydrogenase from cell extracts migrates with enzyme purified from adult chicken liver on SDS/PAGE. Both the native 150-kDa subunit and the 130-kDa form of the enzyme is observed. N-terminal sequence analysis of the 150-kDa subunit shows the following; Ala-Pro-Pro-Glu-Thr-Gly-Asp-Glu-Leu-Val-Phe-Phe-Val-Asn-Gly-Lys-Lys-Val- Val which is similar to the published N-terminal sequences of rat, mouse and insect xanthine dehydrogenases. Autoradiography of denaturing gels of xanthine dehydrogenase isolated from 32P(i)-labeled hepatocytes demonstrates that the 150-kDa and the 130-kDa forms of the enzyme are phosphorylated. Chemical phosphate analysis of acid-precipitated, electrophoretically pure chicken liver xanthine dehydrogenase also shows the presence of covalently bound phosphate. Phosphoamino acid analysis of both 32P-labeled forms of the enzyme demonstrates the presence of phosphoserine. Thus, chicken liver xanthine dehydrogenase contains a phosphoserine residue as found previously in bovine milk xanthine oxidase [Davis, M. D., Edmondson, D. E. & Müller, F. (1984) Eur. J. Biochem. 145, 237-250].

Amino Acid Sequence↗

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↗

Effects of chlorobenzenes on hepatic porphyrin and drug metabolism in chick embryo and day-old chick.

Administration of monochlorobenzene (MCB), p-dichlorobenzene (DCB), or 1,2,4-trichlorobenzene (TCB) at single doses of 800 mg/kg produced an increase in the total porphyrin content of liver of day-old chicks. Porphyrinogenic activity was higher in DCB- and TCB-treated chicks than in MCB-treated chicks. Administration of any of these compounds to chick embryos (40 mg/egg) failed to produce an induction of liver porphyrins. TCB enhanced the excretion of porphyrin in bile of chicks but not of chick embryos. In day-old chicks TCB increased hepatic cytochrome P-450 levels and the activity of 7-ethoxyresorufin deethylase. However, 7-ethoxycoumarin deethylase activity was decreased while cytosolic glutathione S-transferase activity was unchanged. These results point out the differential response of chick embryos and day-old chicks to chlorobenzene-induced changes in porphyrin metabolism as well as the differential induction of microsomal monooxygenases in chicks by TCB.

Animals↗

Actin organization in chick embryo fibroblasts after influenza virus infection. I. Isolation and characterization of actin from chick embryo cells.

Comparison of two starting materials for actin purification has shown that preparation of actin from aceton-dried cytoskeleton was more effective than from native chick embryos (CE). The isolated actin formed a single band of Mr = 42-43000 in SDS-PAGE; less purified samples revealed additional faint bands. G form of actin (non-polymerized) inhibited the activity of DNase I, electron microscopy showed actin filaments and bundles formed upon its polymerization. The freshly purified homogeneous actin has not lost its DNase I-inhibiting activity when incubated for 60 min at 35 degrees or 45 degrees C. Older or less purified actin samples kept under similar conditions showed 18-25% decrease of their DNase I-inhibiting activity and a loss of their polymerization ability. Digestion with trypsin caused a decrease of DNase I-inhibiting activity of fresh as well as for older actin samples.

Acetone↗

Cultivation of trematodes in chick embryos.

The chick embryo can be used as a substitute for adult animals in the cultivation of a variety of parasites. Bernard Fried describes its use in cultivating trematodes, and suggests that the system might be useful as a preliminary chemotherapeutic screen.

Journal Article↗