Biomedical subjects

L H Defize

Publications and source records attributed to L H Defize.

42 records · Page 3Linked to original sources

PubMed1986

Ionic signalling by growth factor receptors.

The proliferation of cells in vivo and in culture is regulated by polypeptide growth factors, such as epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Growth factors initiate their action by binding to specific cell surface receptors. Receptor occupancy triggers a cascade of physiological changes in the target cell which ultimately lead to DNA synthesis and cell division. Immediate consequences of receptor activation include tyrosine-specific protein phosphorylations, a sustained increase in cytoplasmic pH (pHi) and a transient rise in free Ca2+. The rise in pHi has a permissive effect on DNA synthesis and is mediated by an otherwise quiescent Na+/H+ exchange mechanism in the plasma membrane, which is turned on by protein kinase C, the cellular receptor for phorbol esters. The rapid Ca2+ signal is due to either release from internal stores (PDGF) or net entry via a voltage-independent channel in the plasma membrane (EGF). Phorbol esters, acting via kinase C, inhibit the growth factor-induced Ca2+ signals without affecting resting Ca2+ levels. Monoclonal antibodies against the human EGF receptor can act as partial agonists in that they activate the tyrosine-specific protein kinase without inducing any of the ionic signals. These antibodies fail to induce DNA synthesis when added to quiescent fibroblasts, indicating that the Ca2+ and pHi signals can be dissociated from tyrosine kinase activity and suggesting that these signals are indispensable for the stimulation of cell proliferation.

Animals↗

PubMed1986

Ontogeny of the 35K epsilon crystallin during Rana temporaria lens development.

Epsilon is a recently described eye lens protein from Rana temporaria, an anuran amphibian. It is oligomeric with a subunit M.W. of 35K. The cDNA coding for 35K E- in frog lens does not show any homology with cDNA's coding for alpha-, beta-, gamma-, and delta-crystallins. Immunologically, it also does not react with antibodies directed against alpha-, beta-, and gamma-crystallins. The ontogeny of this 35K E-protein has been investigated in R. temporaria lens development by the indirect immunofluorescence staining method with an antibody specific for the 35K E-protein. The purity of the isolated 35K protein and the specificity of the antibody were controlled by Tris-SDS gel electrophoresis and immuno-blotting, respectively. The first positive immunofluorescence reaction was observed in the inner cell wall of a stage V lens. In the external layer/epithelium the reaction was first detected in a single cell of a stage VII lens. Additional positive cells in the external layer/epithelium were detected at an early state VIII and the reaction appeared to be patchy. This type of patchy reaction was also observed in the epithelium of froglet (sub-adult) eye lens.

Animals↗

PubMed1985

Ontogeny of the 38K epsilon-polypeptide during lens development of the duck Anas platyrhynchos.

The epsilon-crystallin polypeptide is a recently described protein in the eye lens of the peking duck, Anas platyrhynchos. It does not cross react with alpha-, beta-, delta-, and gamma-crystallins. It has a molecular weight of 120K and consists of 3 identical 38K polypeptides. It is found in some reptiles and birds. The ontogeny of this polypeptide has been investigated in the developing A. platyrhynchos lens via the indirect immunofluorescence staining using a homologous antibody. The 38K polypeptide was extracted from 13% Tris-SDS acrylamide gels, lyophilized and injected into a young rabbit to raise an antibody. The purity of the isolated 38K polypeptide and and the specificity of the antibody were checked by Tris-SDS gel electrophoresis and immunoblotting, respectively. The first positive reaction is detected at 80 h (stage 18) incubated lens. It is confined to a few elongating early primary fibres. Until the 9th day of development the reaction is confined to the primary and secondary lens fibres. The first positive reaction in the annular pad area is observed in the "day 10" lens. In the anterior epithelium the first positive reaction is detectable in the "day 12" lens. At the beginning it is confined to a few cells in the center of the epithelium and gradually the reaction spreads to other cells. A strong and uniform reaction in the entire epithelium is noted for the first time in the lens of a just-hatched duckling. The 38K epsilon-polypeptide is detectable after the alpha-, beta-, and delta-crystallins, which, in the duck, appear simultaneously from 66 h (stage 15/16).

Aging↗

PubMed1985

Visualization of epidermal growth factor receptor in cryosections of cultured A431 cells by immuno-gold labeling.

Cryo-ultramicrotomy in combination with immuno-gold labeling has been demonstrated to present a powerful tool in the visualization of extra- and intracellular located antigens. We have applied this method to localize epidermal growth factor (EGF) receptor in cultured A431 human epidermoid carcinoma cells. However, both the labeling efficiency, maintenance of antigenicity, and the recognizability of the ultrastructure in cryosections are highly dependent upon the fixation procedures. Using 125I-EGF or a consecutive labeling with a monoclonal anti EGF-receptor antibody, rabbit-anti-mouse antibody and 125I-protein A, it was shown that maintenance of antigenicity was optimal using 2% paraformaldehyde as a fixative, whereas under these conditions also the recognizability of ultrastructure was sufficient. After appropriate fixation and labeling, gold particles were observed associated with various regions of the plasma membrane, including coated pits, and with various types of vesicles, including coated vesicles, intracellular vesicular membranes, multi-vesicular bodies and lysosomes. The results indicate that this method allows a visualization of EGF-receptors and resolution of the EGF-receptor processing pathway at the electron microscopic level, independent of the internalization process of labeled ligands.

Carcinoma, Squamous Cell↗

PubMed1985

Ionic signal transduction in growth factor action.

Growth factors are polypeptides which exert their mitogenic action through binding to specific high-affinity receptor molecules on the cell surface of target cells. This interaction leads to the rapid activation of a receptor-linked signal transduction system, involving the stimulation of an intrinsic receptor tyrosine phosphokinase activity, the breakdown of inositol lipids, and the production of ionic signals. In this contribution we have analysed the nature and origin of the ionic signals, and we have applied monoclonal antibodies against the receptor for epidermal growth factor (EGF), as well as tumour-promoting phorbol esters, to dissociate the early cellular responses to growth factors. Evidence is presented that the ionic signals are coupled to the breakdown of inositol lipids. The hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) would lead to the production of 1,2-diacylglycerol (DG) and inositol triphosphate (IP3). DG production results in the stimulation of protein kinase C, which causes the activation of Na+/H+ exchange by increasing its affinity for cytoplasmic H+. Consequently, a rise in cytoplasmic pH is observed. This response can be mimicked by the tumour promoter TPA, which can replace DG in activating the protein kinase C. Independently, IP3 production leads to the rapid mobilization of Ca2+ from intracellular stores. Monoclonal antibodies against the EGF receptor differed in their ability to evoke EGF-like responses upon binding to the EGF receptor. Of the three anti-EGF receptor IgGs tested, one was directed against the EGF-binding domain (2E9), and the others (2D11 and 2G5) were directed against sugar moieties not involved in EGF binding. Receptor tyrosine kinase activity could be stimulated by 2E9 as well as 2D11, but not by 2G5. Only 2D11 induced morphological changes similar to EGF. None of the antibodies was able to trigger the production of ionic signals, which implies probably that antibody binding to the receptor, even when they bind to the EGF binding domain, is an insufficient stimulus for the breakdown of inositol lipids. Most importantly, these monoclonal antibodies were also not able to induce DNA synthesis in quiescent human fibroblasts, not even after cross-linking of the EGF receptors by a second antibody. It may thus be concluded that the stimulation of the intrinsic receptor tyrosine phosphokinase activity can be dissociated from other early responses, and that none of the identified early responses is a sufficient trigger for the mitogenic action of growth factors.

Animals↗

PubMed1982

Transfer properties of the bovine brain phospholipid transfer protein. Specificity towards phosphatidylcholine analogs and the inhibitory effect of sphingomyelin.

A coupled transport of phosphatidylinositol from the monolayer to phosphatidylcholine vesicles, and a phosphatidylcholine transport in the reverse direction in the presence of bovine brain transfer protein is demonstrated. No significant amounts of protein accumulate at the interface during the transfer reaction. The transfer protein from bovine brain shows a lower specificity for phosphatidylcholine than does the transfer protein from bovine liver. Relative to egg phosphatidylcholine a low transfer rate is found for derivatives with a chain length of 14 carbon atoms and a distance between phosphorus and nitrogen of 6 carbon atoms. The gel state of phosphatidylcholine does not reduce the transfer reaction as catalyzed by the bovine brain protein. The transfer of phosphatidylinositol is inhibited by sphingomyelin. The presence of 200 mM K+ or 1mM Ca2+ does not affect the transfer activity of the bovine brain protein. Divalent ions at concentrations higher than 5 mM cause a fusion of vesicles with monolayers. The pH optimum of the phosphatidylinositol transfer reaction is 8.

Animals↗