Amidolytic activity of 7S nerve growth factor (original) (raw)
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The Journal of Comparative Neurology, 1994
Touch domes are clearly delineated mechanoreceptors that are visible on the depilated skin of mammals. These structures consist of a sharply circumscribed disk of thickened epithelium surmounting a group of Merkel cells that are innervated by type I sensory neurons. These characteristic cutaneous structures provide an ideal opportunity for investigating whether the localization of nerve growth factor (NGF) in the skin is related to sites of sensory axon termination. For these reasons, we have used immunocytochemistry to study the distribution of NGF and the low-affinity NGF receptor (~7 5~~~~) in the touch domes of adult rat skin. Intense NGF-like immunoreactivity was sharply restricted to keratinocytes (excluding the stratum corneum) of the thickened epidermis of touch domes. The epidermis immediately surrounding touch domes and the epidermis of the tylotrich hair follicle associated with touch domes were not stained by anti-NGF antiserum. Merkel cells of the basal epidermis of touch domes were immunonegative for NGF but were immunopositive for p7FiNGFR as were the type I nerve endings innervating these cells. Quantitative Northern blotting revealed that the level of NGF mRNA was substantially higher in keratinocytes isolated from the stratum granulosum and stratum spinosum than in keratinocytes isolated from the stratum germinativum. These findings indicate that NGF synthesis in mature skin has a highly restricted regional distribution that is primarily associated with the innervation of a specialized touch receptor.
Developmental Biology, 1997
Embryonic mouse skin undergoes a drastic morphological change from 13 to 16 gestational days, i.e., formation of rudiments of hair follicles and stratification and cornification of interfollicular epidermis. To investigate underlying molecular mechanisms of the morphogenesis, we established an organ culture system that allows skin tissues isolated from 12.5-or 13.5-days postcoitus embryos to develop in a manner that is histologically and temporally similar to the process in vivo. Expression of differentiation markers of epidermal keratinocytes including cholesterol sulfotransferase and cytokeratin K1 was induced in culture, as it occurs also in vivo. The morphogenic process was observed by time-lapse videomicrography. In this culture system, epidermal growth factor (EGF) and transforming growth factor a specifically and completely inhibited the hair follicle formation with marginal effects on interfollicular epidermis. The inhibitory action by EGF was reversible and stage specific, i.e., at an early stage of the development of hair rudiments. Among known ligands to the EGF receptor, Schwannoma-derived growth factor and heparin-binding EGF were expressed in in vivo epidermis during the period of the initial formation of hair follicles. EGF receptor is expressed in epidermis throughout the developing period examined. Using an adenovirus vector, we demonstrated that the lacZ gene was transduced into the epidermal and dermal cell layers without appreciable toxicity. These results indicate that the present culture system provides a unique opportunity to investigate molecular mechanisms of skin morphogenesis including the role of EGF signaling under defined experimental conditions.
Archives of Biochemistry and Biophysics, 1974
Epidermal growth factor, a 6,400-dalton polypeptide from the mouse submaxillary gland, binds specifically to cells and membranes derived from a variety of human, rat, mouse, and bovine tissues. Liver, placenta, skin, cornea, and cultured chondrocytes, Hela cells, and Chang liver cells bind large amounts of epidermal growth factor, whereas fat cells, resting and lectin-stimulated human peripheral lymphocytes, mouse thymocytes, cultured rat hepatoma cells, and mammary cells from virgin and pregnant mice bind little or no epidermal growth factor. The binding site for epidermal growth factor is distinct from receptors for other anabolic peptides such as insulin, nerve growth factor, and growth hormone. The binding of epidermal growth factor is rapid and reversible. The rate constant of association is approximately 10" mole-' set-I, the rate constant of dissociation is about 6 x lo-' set-', and the apparent equilibrium dissociation constant is about lode M. Trypsin at low concentrations (50-200 pg/ml) destroys the receptor site for epidermal growth factor. The binding of epidermal growth factor by membranes is not accompanied by appreciable degradation of the peptide present in the medium or of that bound to the membranes. Use can be made of the high affinity and specificity of membranes for epidermal growth factor to measure by a competitive binding assay as little as 200 pg of EGF per ml (3 x lo-" M). Epidermal growth factor (EGF)6, a 6,400-dalton disulfide-containing polypep
Journal of molecular neuroscience : MN, 2014
Nerve growth factor (NGF) treatment causes a profound down-regulation of epidermal growth factor (EGF) receptors (EGFR) during the neuronal differentiation of PC12 cells. This process was characterized by a progressive decrease in EGFR level, as measured by (125)I-EGF binding and Scatchard analysis, tyrosine phosphorylation, Western blotting, and bio-imaging using EGF-labeled with a near-infrared probe. Differentiation of the cells with NGF for 5-7 days produces a 95 % reduction in the amount of (35)S-methionine-labeled EGFR. This down-regulation does not occur in PC12-nnr5 cells, which lack the TrkA NGF receptor but is reconstituted in these cells upon their stable transfection with TrkA. The process of NGF-induced EGFR down-regulation was inhibited by K252a, a TrkA antagonist and by anti-TrkA antibodies but not by Thx-B, a blocker of the interaction of NGF with p75(NTR) receptors. NGF-induced (heterologous) down-regulation, but not EGF-induced (homologous) down-regulation of EGFR,...
The International Journal of Developmental Biology
DECM macromolecules create a specific environment that participates in the control of cell proliferation and differentiation during embryogenesis. Quantitative and qualitative alterations in the ECM may depend on several growth factors that modify cell metabolism. Since transforming growth factor β (TGFβ) and α (TGFα) are abundantly expressed during embryonic development in organs in which epithelial-mesenchymal interactions occur, the aim of this study was to determine: a) the effect of TGFβ on the phenotype of 7 and 14 day chick embryo back skin (CEBS) fibroblasts by evaluating the neosynthesis of GAG, collagen and fibronectin; b) whether TGFα and TGFβ production, in particular TGFβ 3 and TGFβ 4 , and the number of TGFβ receptors change during these two stages of embryonic development. The results show that the neosynthesis of ECM macromolecules, tested using radiolabelled precursors, is increased by TGFβ. The growth factor generally favours cellular accumulation more than secretion. As far as GAG is concerned, TGFβ has a greater stimulatory effect on sulphated GAG than on HA. Specific bioassay shows that TGFβ 3 and TGFβ 4 activity is higher in 7 day than 14 day CEBS fibroblasts. Moreover, TGFβ 3 and TGFβ 4 mRNA expression is increased in the first stages of development. Instead, the level of TGFα increases in successive developmental stages. Since TGFα stimulates the synthesis and secretion of HA, and HA binds and inactivates TGFβ, the greater quantity of HA in 14 day fibroblasts may contribute to reducing the TGFβ effect. Overall our data suggest that the production of TGFβ and TGFα are agedependent and that the balance between the two growth factors may be a mechanism for controlling skin differentiation.