Changes in lectin-induced deoxyribonucleic acid synthesis in cultures of chick-embryo fibroblasts at various stages of development.
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ABSTRACT:
SUBMITTER: Roguet R
PROVIDER: S-EPMC1172487 | biostudies-other | 1975 Nov
REPOSITORIES: biostudies-other
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Project description:The toxic effect of Ricinus lectin RCA I, as estimated by the inhibition of [3H]leucine incorporation, was investigated on chick-embryo fibroblasts at different stages of development. There appeared to be a differential susceptibility of chick-embryo fibroblasts to lectin RCA I. Fibroblasts from 16-day embryos were the most sensitive to its toxic effect in terms of both concentration and time, and cells from 8-day embryos were the least sensitive. This differential sensitivity to the toxic effect of lectin RCA I was closely related to the binding of the lectin: fibroblasts from 16-day embryos had more binding sites (1.5 x 10(7)/cell) with a high affinity than did 12-day (0.45 x 10(7)/cell) or 8-day embryos (0.2 x 10(7)/cell). Studies on the specificity and the removal of bound lectin RCA I by D-galactose indicated that the lectin binding was necessary but not sufficient in itself to cause the toxic effect and that the lectin needed to enter the cells in order to be toxic. The amount of lectin RCA I needed to induce a 50-60% toxicity enters fibroblasts of 16-day embryos more rapidly than those of 12- and 8-day embryos.
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Project description:Nerve growth factor (NGF) was discovered because of its neurotrophic actions on sympathetic and sensory neurons in the developing chicken embryo. NGF was subsequently found to influence and regulate the function of many neuronal and non neuronal cells in adult organisms. Little is known, however, about the possible actions of NGF during early embryonic stages. However, mRNAs encoding for NGF and its receptors TrkA and p75(NTR) are expressed at very early stages of avian embryo development, before the nervous system is formed. The question, therefore, arises as to what might be the functions of NGF in early chicken embryo development, before its well-established actions on the developing sympathetic and sensory neurons. To investigate possible roles of NGF in the earliest stages of development, stage HH 11-12 chicken embryos were injected with an anti-NGF antibody (mAb ?D11) that binds mature NGF with high affinity. Treatment with anti-NGF, but not with a control antibody, led to a dose-dependent inversion of the direction of axial rotation. This effect of altered rotation after anti NGF injection was associated with an increased cell death in somites. Concurrently, a microarray mRNA expression analysis revealed that NGF neutralization affects the expression of genes linked to the regulation of development or cell proliferation. These results reveal a role for NGF in early chicken embryo development and, in particular, in the regulation of somite survival and axial rotation, a crucial developmental process linked to left-right asymmetry specification.
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Project description:1. The liver of the domestric fowl (Gallus gallus) remains capable of conjugating o-aminophenol with glucuronic acid after 8 days' culture. The pathway of o-aminophenyl glucuronide formation in cultured liver, as in fresh tissue, includes the enzyme UDP-glucuronyltransferase. 2. UDP-glucuronyltransferase activity in chick-embryo liver increases on culture from very low to adult values within 6-8 days. 3. The development of UDP-glucuronyltransferase activity in cultured chick-embryo liver requires certain serum factors in the medium. The requirements change with embryo age. Liver from embryos younger than 15 days develops enzyme activity equally well in media containing either foetal or adult serum; liver from embryos older than 16 days develops activity only with adult serum. The development of enzyme activity in liver from the older embryos appears to be stimulated by diffusible factors in adult serum and inhibited by diffusible factors in foetal serum. It is suggested that the stimulation and inhibition of enzyme formation by small, diffusible molecules may be part of the mechanism regulating UDP-glucuronyltransferase activity in vivo. 4. Liver from 19-day-old chick embryos cultured with foetal serum begins to develop UDP-glucuronyltransferase activity if transferred to an adult-serum medium. Its capacity to develop UDP-glucuronyltransferase activity in adult serum survives in a foetal-serum medium for at least 5 days, the longest period tested. 5. The activity of UDP-glucuronyltransferase reached in 19-day chick-embryo liver after 1 or 2 days with adult serum is maintained without further increase after transfer to a foetal-serum medium. After 3 days with adult serum UDP-glucuronyltransferase activity continues to increase when the tissue is transferred to a foetal-serum medium. Thus liver from 19-day-old embryos requires 3 days with adult serum before development of enzyme activity becomes independent of a continuous adult-serum environment.
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Project description:1. The ability of N- and C-terminal half-molecule fragments of hen ovotransferrin to interact with chick red blood cells (CERBC) has been studied under conditions that allow binding of the transferrin to transferrin receptors to take place, but not the delivery of iron to the cell. Two kinds of half-molecule fragments were used: (a) those which can associate with one another to give a dimer resembling native transferrin and (b) those which cannot associate in this way because they lack a few amino acid residues from their C-terminal ends. 2. Neither N nor C half-molecules alone can bind to the CERBC, but, when both are present, tight binding occurs. 3. Whether or not the half-molecules can associate with one another makes little difference to receptor binding. 4. Given that one of the half-molecules is iron-saturated, the presence or absence of iron in the contralateral half-molecule again makes little difference to receptor binding.
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Project description:The embryonic chick occupies a privileged place among animal models used in developmental studies. Its rapid development and accessibility for visualization and experimental manipulation are just some of the characteristics that have made it a vertebrate model of choice for more than two millennia. Until a few years ago, the inability to perform genetic manipulations constituted a major drawback of this system. However, the completion of the chicken genome project and the development of techniques to manipulate gene expression have allowed this classic animal model to enter the molecular age. Such techniques, combined with the embryological manipulations that this system is well known for, provide a unique toolkit to study the genetic basis of neural development. A major advantage of these approaches is that they permit targeted gene misexpression with extremely high spatiotemporal resolution and over a large range of developmental stages, allowing functional analysis at a level, speed and ease that is difficult to achieve in other systems. This article provides a general overview of the chick as a developmental model focusing more specifically on its application to the study of eye development. Special emphasis is given to the state of the art of the techniques that have made gene gain- and loss-of-function studies in this model a reality. In addition, we discuss some methodological considerations derived from our own experience that we believe will be beneficial to researchers working with this system.
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Project description:A chick-embryo fibroblast lambda gt11 cDNA library was screened with affinity-purified antibodies to chick gizzard vinculin. One recombinant was purified to homogeneity and the fusion protein expressed in Escherichia coli strain C600. The fusion protein was unstable, but polypeptides that reacted with vinculin antibodies, but not non-immune immunoglobulin, were detected by Western blotting. The recombinant contained a single EcoRI fragment of 2891 bp with a single open reading frame. The deduced protein sequence could be aligned with that of six CNBr-cleavage peptides and two tryptic peptides derived from chicken gizzard vinculin. AUG-247 has tentatively been identified as the initiation codon, as it is contained within the consensus sequence for initiation sites of higher eukaryotes. The cDNA lacks 3' sequence and encodes 74% of the vinculin sequence, presuming the molecular mass of vinculin to be 130,000 Da. Analysis of the deduced sequence showed no homologies with other protein sequences, but it does display a triple internal repeat of 112 amino acid residues covering residues 259-589. The sequences surrounding the seven tyrosine residues in the available sequence were aligned with the tyrosine autophosphorylation consensus sequence found in protein tyrosine kinases. Tyr-822 showed a good match to this consensus, and may represent one of the two major sites of tyrosine phosphorylation by pp60v-sre. Northern blots showed that the 2.89 kb vinculin cDNA hybridized to one size of mRNA (approx. 7 kb) in chick-embryo fibroblasts, chick smooth muscle and chick skeletal muscle. Southern blots revealed multiple hybridizing bands in genomic DNA.
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