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Deoxyguanylate when added to chick embryos grown in explant inhibited growth and development. Deoxycytidylate deaminase activity was inhibited both in the explants and in vitro; since the effect was quite specific, it is suggested that this may represent another control mechanism for deoxynucleotide synthesis.
In vitro assays for the beta-N-acetylglucosaminyltransferases (GlcNAcTase) were performed on crude microsomal fractions prepared from 8-day chick embryo fibroblasts (8-day-CEF) and 16-day chick embryo fibroblasts (16-day-CEF) using [3H]mannose-labeled GlcNAc beta 1----2 Man alpha 1----6 (GlcNAc beta 1----2 Man alpha 1----3) Man beta 1----4 GlcNAc beta 1----4 (Fuc alpha 1----6) GlcNAc-Asn and UDP-GlcNAc as substrates. 8-day-CEF synthesize preferentially triantennary complex type chains, whereas 16-day-CEF produce essentially tetraantennary complex type chains. Furthermore oligosaccharides containing the GlcNAc beta 1----4 Man alpha 1----3 Man sequence represent 90% of the structures found in 16-day-CEF versus 30% in 8-day-CEF, indicating an increase in beta-N-acetylglucosaminyltransferase IV activity during embryo development.
Acetyl-CoA carboxylase catalyzes the first committed step in the synthesis of fatty acids. Because fatty acids are required during myelination in the developing brain, it was proposed that the level of acetyl-CoA carboxylase may be highest in embryonic brain. The presence of acetyl-CoA carboxylase activity was detected in chick embryo brain. Its activity varied with age, showing a peak in the 17-18-day-old embryo and decreasing thereafter. The enzyme, affinity-purified from 18-day-old chick embryo brain, appeared as a major protein band on polyacrylamide electrophoresis gels in the presence of sodium dodecyl sulfate (Mr 265,000), indistinguishable from the 265 kDa isozyme of liver acetyl-CoA carboxylase. It had significant activity (Sp act = 1.1 mumol/min per mg protein) in the absence of citrate. There was a maximum stimulation of only 25% in the presence of citrate. Dephosphorylation using [acetyl-CoA carboxylase] phosphatase 2 did not result in activation of the enzyme. Palmitoyl-CoA (0.1 mM) and malonyl-CoA (1 mM) inhibited the activity to 95% and 71%, respectively. Palmitoylcarnitine, however, did not show significant inhibition. The enzyme was inhibited (greater than 95%) by avidin; however, avidin did not show significant inhibition in the presence of excess biotin. The enzyme was also inhibited (greater than 90%) by antibodies against liver acetyl-CoA carboxylase. An immunoblot or avidin-blot detected only one protein band (Mr 265,000) in preparations from chick embryo brain or adult liver. These observations suggest that acetyl-CoA carboxylase is present in embryonic brain and that the enzyme appears to be similar to the 265 kDa isozyme of liver.
Acetazolamide was injected into chick embryos on the 14th or 15th day of incubation. Doses ranging between 5 and 10 mg per egg produced a retardation in the growth of long bones. The affected bones contained a normal proportion of mineral as determined by ashing and presented a normal histological picture. On the basis of these findings, it is suggested that the alterations were not due to a specific direct effect of the drug on bones. The incorporation of 131-I by the thyroid glands of acetazolamide-injected embryos was analyzed radioautographically and quantitated on the same 6 mu-paraffin sections, with a thin window Geiger counter. The incorporation appeared notably reduced 3 h after the injection of acetazolamide and the reduction persisted for a least 24 h.the electron microscopical observation of thyroid follicular cells from similarly treated embryos showed that the cytological characteristics indicating an active protein synthesis were unmodified with respect to those found in control embryos. These results may indicate that acetazolamide inhibits the iodination of the throid hormone without interfering with the synthesis of the globulin. It is suggested that the growth retardation observed in the embryos treated with acetazolamide may be secondary to the action of the drug on the thyroid gland, although this action appears to be a transitory one.
The liver plays a crucial role in metabolism. There is considerable interest in how the liver develops, as such knowledge could prove of importance in regenerative medicine. However, our understanding of liver development remains somewhat limited. We have developed a model system using the chick embryo that is cost effective and is easy to manipulate experimentally. We performed four fundamental studies: (i) construction of an atlas of the developing chick liver; (ii) identification of differentiation marker genes in the developing chick embryo; (iii) development of germ-layer specific electroporation; and (iv) establishment of organ culture from the developing chick liver. Using this system, we have been able to demonstrate the functions of candidate genes within a shorter period and in a more cost-effective manner. In parallel with the establishment of this system, we examined the expression patterns of genes known to be required for organ development in the developing chick embryo in order to identify genes also involved in liver development. To date, we have found sixteen genes that are expressed in the developing chick liver (GELD, genes expressed in liver development). This knowledge will be fundamental to the establishment of the basic technology for engineering liver tissue in the future.
The effects of minoxidil were studied on chick embryos of 24 and 48 hours of incubation. Minoxidil (3%) was injected into the air sacs of the eggs at doses of 20, 30, 40, and 50 microliters per egg. The controls received 100 microliters of physiological saline. All the embryos, including controls, were examined at day 13. The total number of eggs used in this study was 300. At 24 hours incubation, the percentage of survival ranged from 87 to 21 as the dosages of minoxidil were increased from 20 microliters to 50 microliters per egg (controls = 87%). The survival of the embryos ranged from 79% to 9% after the 48-hour treatment with the similar dosages of minoxidil utilized for the 24-hour group (controls = 83%). A low incidence of gross malformations such as twisted limbs, abnormal beak, short neck and everted viscera were observed; however, the increased incidence was not statistically significant when compared to controls. Body hemorrhage and edema were of high occurrence among the treated embryos. These effects are probably secondary to the known pharmacological effects of minoxidil. The frequency and types of gross malformations did not vary much in the 24 or 48-hour treated groups.
42 hour-old chick embryos are operated by microsurgical means. The encephalon, which is normally closed at this stage, is reopened and the incision extended to the level of 2nd or 3rd somites. The experimental embryos are anencephalic. Their comparison with a spontaneous anencephalus shows a large morphological similarity. These first experiments confirm the hypothesis by which anencephaly is a direct consequence of the failure of neural tube closure.
The effect of the lighting schedule on the pharmacological action of theophylline was studied in chick embryos. Fertile eggs of White Leghorns were incubated and investigated, on two occasions, under constant light conditions or under constant dark conditions. A single injection of theophylline 2.14, 4.29, 8.57 and 17.14 mg/egg into the air sac of fertile eggs was carried out on the 16th day of incubation. Electrocardiograms (ECGs) were recorded 0 to 60 min after drug injection. After drug injection, heart rate increased under constant light conditions, but decreased under dark conditions. In addition, arrhythmia was produced by theophylline, 17.14 mg/egg, under constant dark conditions. These results indicate that the manipulation of the lighting schedule may have a marked influence on the pharmacological effects of theophylline in chick embryos.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), beta-naphthoflavone (beta NF), and phenobarbital (PB) cause marked induction of cytochrome P-450 (P-450)-mediated arachidonic acid metabolism in chick embryo liver. We show here that the P-450 arachidonic acid epoxygenases induced by TCDD and beta NF are immunochemically indistinguishable from each other and unrelated to the arachidonic acid epoxygenase induced by PB. On Western blots, IgG from an antiserum against beta NFAA, a 55-kDa P-450 arachidonic acid epoxygenase purified from beta NF-treated chick embryo liver, immunoreacted selectively and to the same extent with a 55-kDa band in liver microsomes from chick embryos treated with TCDD or beta NF. It failed to react with proteins from untreated, solvent-treated, or PB-treated embryos on immunoblots or to immunoinhibit PB-induced arachidonic acid metabolism. Anti-beta NFAA IgG immunoinhibited all arachidonic acid metabolism by reconstituted beta NFAA and formation of arachidonic epoxides (EETs) and monohydroxylated derivatives (HETEs) by microsomes from TCDD- and beta NF-treated livers; it did not inhibit omega-hydroxylation. In contrast, IgG from an antiserum against the major PB-induced chicken P-450s, 2H1 and 2H2, immunoreacted with two major PB-induced P-450s, of 48 and 49 kDa, on Western blots. It also immunoinhibited formation of EETs and HETEs by PB-treated microsomes entirely and omega-hydroxylation by 50%. It failed to react with TCDD- or beta NF-induced P-450s on Western blots or to immunoinhibit TCDD- or beta NF-induced arachidonic acid metabolism. Because other P-450s with which anti-beta NFAA and anti-PB IgG cross-reacted were inactive in arachidonic acid epoxygenation, the findings are consistent with beta NFAA being principally responsible for the epoxygenation induced by TCDD and beta NF and 2H1 and/or 2H2 being responsible for epoxygenation induced by PB. Further, the P-450 arachidonate omega-hydroxylase and the epoxygenase in livers of TCDD- or beta NF-treated embryos are immunochemically unrelated, whereas those in livers of PB-treated embryos may be partly related.
Treatment with neuraminidase (100 units/ml) of chick embryo fibroblasts in vitro only partially inhibits adsorption of fowl plague virus on these cells. Cultivation of chick embryo fibroblasts in the presence of 50 units/ml neuraminidase had no effect on the sensitivity of these cells to fowl plague virus and on the extent of virus reproductions. It is suggested that neuraminic acid which is a component of the external cell membrane is not only substance responsible for adsorption of orthomyxoviruses.
The study described below was performed as a continuation of a previous study in which we found reduced motoneuron number in lumbar spinal cord of the chick embryo following chronic ethanol administration from embryonic day 4 (E4) to E11. We sought to determine whether this reduction was due to primary ethanol toxicity or to enhancement of naturally occurring cell death (NOCD) and to determine whether administration of ethanol at a later period of development could also reduce motoneuron number. Earlier studies have shown that curare suspends NOCD in the chick embryo. By administering both ethanol and curare to these embryos from E4 to E11 and examining the lumbar spinal cord on E12, we determined that ethanol was directly toxic to motoneurons and reduced motoneuron number in the absence of NOCD. By administering ethanol from E10 to E15 and examining the lumbar spinal cord on E16, we determined that ethanol can reduce motoneuron number without altering spinal cord length during more than one stage of chick embryo development, and that ethanol toxicity is not dependent on NOCD. In addition, we demonstrated that ethanol does not affect the neurotrophic content of chick muscle when it is administered from E10 to E15.
The effects of chloramphenicol (CAP) and thiamphenicol (TAP) on the outcome of Chlamydia psittaci infection in chick embryos were compared. CAP administered along with Chlamydia reduced embryo mortality rates but showed no appreciable effects when its injection was delayed. On the contrary, TAP caused a high rate of embryo survival in both experimental situations. Statistical analysis of the results showed that differences in the survival rates following CAP and TAP administration are significant. Metabolic pathways in chick embryos of the antibiotics assayed differed remarkably in that CAP undergoes a quicker inactivation, which could even justify the better activity showed by TAP.
Primary cultures of chick embryo hepatocytes are known to maintain their initial level of cytochrome P-450 for a number of days. To explore the possibilities of chick embryo hepatocyte cultures as a tool in drug metabolism, induction profiles of cytochrome P-450 were determined and the metabolism of benzo(a)pyrene as a model substrate was studied. Maximum induction by phenobarbitone and Aroclor 1254 is reached after 21 h and 18 h, respectively, both in the presence and absence of serum. For beta-naphthoflavone induction is maximal after 31 h in the presence and 43 h in the absence of serum. The levels of P-450 after induction are comparable to those found in vivo in rats: increases of 200% for phenobarbitone, 200% for beta-naphthoflavone and 210% for Aroclor 1254. Ethoxyresorufin-O-deethylase activities are induced by beta-naphthoflavone and Aroclor 1254, but as expected only slightly by phenobarbitone. In the absence of serum in the culture medium, for the control as well as the induced cells a plateau of activity is maintained for at least 24 h. In the presence of serum a decline in P-450 levels is observed. Especially in the case of Aroclor, an increase in porphyrin content of 320% of control values is seen at the same time. A number of representative metabolites of benzo(a)pyrene were quantitated during a 4-h incubation. Relative amounts are comparable to those observed with rat liver microsomes. As expected, beta-naphthoflavone and Aroclor induce the rate of metabolism (by 500% and 400%, respectively, in the absence of serum), but phenobarbitone has no or very little effect.(ABSTRACT TRUNCATED AT 250 WORDS)
ECGs free of movement artefacts were obtained without anesthesia in 16- to 18-day-old chick embryos close to hatching and used to study the effect of the environmental toxin 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) on cardiac rhythm and conduction. The ECGs of normal late stage chick embryos exhibited short PR intervals, frequent nonisoelectric PR segments, delta waves, and inverted T waves. Those ECG characteristics are found in patients with the Wolff-Parkinson-White syndrome (WPW) in which they reflect ventricular preexcitation associated with the use of accessory conduction pathways and arrhythmias. Isoproterenol (30 microg/egg) did not alter the ECG preexcitation characteristics. Flecainide, a sodium channel blocker used clinically to suppress WPW accessory pathway activity, at 0.5 to 5 mg per egg diminished the preexcitation and caused atrioventricular (AV) block, supporting the use of accessory pathways together with AV-nodal conduction in normal late stage chick embryos. The findings challenge the dogma that accessory pathways are entirely replaced by AV conduction pathways in late fetal development. TCDD, at 1-2 nmol per egg for 48 h, did not affect heart rate, the increase in heart rate by isoproterenol, or the ECG characteristics, suggesting that short-term TCDD treatment did not affect sinus node function or cardiac conduction. The latter results taken together with prior findings indicate that TCDD differentially impairs the inotropic and lusitropic effects but not the chronotropic or dromotropic effects of isoproterenol. In TCDD-treated embryos, flecainide, tested at 5 mg per egg, caused much less inhibition of preexcitation or production of AV block than in the untreated or solvent-treated controls. The resistance to flecainide represents a new TCDD effect consistent with the reported increase of cardiac myocyte [Ca(2+)](i) by TCDD treatment.
Transverse, frontal and sagittal notochord sections of chick embryos at different developmental stages were ultrastructurally examined. The notochord tissue organization appears to be more likely to build a stiff but flexible body axis than to have an inducting role on the surrounding embryonic structures.
Studies have been made on the activity of glycosidases from eye tissues of developing chick embryos and adult hens. The enzymes of carbohydrates metabolism (hyaluronidase, beta-glycosidase and beta-galactosidase) from the sclera, cornea and ciliary body were examined. It was demonstrated that the distribution of glycosidases in different tissues of the eye is not identical. The activity of beta-glycosidase and beta-galactosidase in all the tissues of 14-day embryos is higher than in adult hens; sharp reduction of the activity was observed at the stage of eye opening. The activity of hyaluronidase in the sclera and cornea of chick embryos is maintained at a low level up to the stage of eye opening, being subjected to minor changes.
The blood flow dynamics in the early chick embryo were visualized by using a video-imaging method without invasion to the circulatory system. The movement of juvenile blood cells in the dorsal aorta was tracked and the flow velocity of blood cells calculated by using an image processor and a computer.
In the present study, we demonstrate that both interleukin-1 (IL-1) and interleukin-6 (IL-6) induced a significant decrease in glycosaminoglycan (GAG) synthesis and, more strikingly, secretion by 7 and 13 day-old chick embryo skin fibroblasts. We demonstrated that interleukin treatment also inhibited the synthesis of collagenase-digestible proteins (type I collagen). In addition, tissue culture supernatants (conditioned media, CM) were tested for reactivity for IL specific ELISAs and for their ability to stimulate proliferative responses in mouse thymocytes and hybridoma cells. Our findings demonstrate that chick embryo skin fibroblasts spontaneously produce IL-1 and, in even greater amounts, IL-6. Highest levels of interleukin secretion were found in the CM of 13 day-old fibroblasts and the IL-1 beta isoform was predominant over IL-1 alpha. Pretreatment of the fibroblasts with either IL-1 or IL-6 increased the secretion of both cytokines. Increased IL-1 levels were correlated with enhanced IL-1 bioactivity in the CM of IL-6 treated fibroblasts. By contrast, the raised concentrations of IL-1 in the CM of IL-1 treated cells and IL-6 in the CM of IL-1 or IL-6 treated fibroblasts failed to translate into augmented bioactivity. These observations, taken together, indicated that IL-1 and IL-6 are able to regulate the synthesis and secretion of ECM macromolecules of developing connective tissues and the cytokine release by chick embryo skin fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)