Biomedical subjects

D Edgar

Publications and source records attributed to D Edgar.

At least 55 records · Page 3Linked to original sources

PubMed1990

Developmental loss of laminin from the interstitial extracellular matrix correlates with decreased laminin gene expression.

The expression of the polypeptide subunits of the glycoprotein laminin in developing mouse tissues was analysed by immunoblots and Northern blots, and by immunohistochemistry at the ultrastructural level. In the neonate, almost all the laminin of the sciatic nerve was freely extractable and was located mainly in the mesenchymal interstitial extracellular matrix, rather than in basement membranes. During the first two postnatal weeks, the distribution of laminin shifted to assume the adult pattern, most being located in basement membranes and insoluble under physiological conditions. Analysis of laminin subunit expression showed that both the mRNA for the laminin B chains and the corresponding polypeptides are widely expressed in nerve and other tissues, the mRNA levels decreasing during the first two postnatal weeks as the amount of laminin in the tissue increased. In contrast, the A chain mRNA and polypeptide were undetectable in nerve at any age studied, although they were present in perinatal kidney and placenta. It is proposed that the large amount of soluble laminin present in the developing interstitial extracellular matrix is a consequence of the high levels of expression of laminin mRNA, the subsequent decrease in expression resulting in the adult distribution where most laminin is insoluble within the basement membrane.

Animals↗

PubMed1989

The high-affinity binding of laminin to cells. Assignation of a major cell-binding site to the long arm of laminin and of a latent cell-binding site to its short arms.

The laminin proteolytic fragments 1 (derived from the intersection of the short arms of the cruciform laminin molecule) and 8 (derived from the laminin long arm) bind to distinct receptors on HT-1080 human fibrosarcoma cells; both fragments are shown here to inhibit the high-affinity binding of laminin to these cells. Inhibition of binding between fragment 8 and laminin was competitive, whereas that between fragment 1 and laminin was noncompetitive. This indicates that laminin and fragment 8 most probably share the same cellular receptors, whereas laminin and fragment 1 bind to distinct receptors, inhibition being due to steric hindrance. Surprisingly, fragment 1-4 (corresponding to the complete short arms of laminin) neither bound to HT-1080 cells nor inhibited the binding of laminin or fragment 1. After treatment of fragment 1-4 with pepsin, however, the smaller subfragment 1 was liberated, which could then bind to the cells, and so was shown to block the binding of laminin and fragment 1. We conclude that native laminin bound to HT-1080 cells via the fragment-8-binding site near the end of its long arm. Although these cells also have distinct receptors for the short arm fragment 1, this receptor-binding site was not used as it appeared to be latent within the native laminin molecule.

Binding Sites↗

PubMed1989

The survival of early chick sympathetic neurons in vitro is dependent on a suitable substrate but independent of NGF.

The neuronal cell population of lumbosacral sympathetic ganglia from 7-day-old chick embryos is characterized by a high proportion of cells with the ability to proliferate in culture (Rohrer and Thoenen, 1987). It is now demonstrated that neither proliferation nor survival of these neurons depend on the presence of nerve growth factor (NGF). However, neuronal survival did depend on the culture substrate used: on laminin, E7 neurons survived and their number increased due to proliferation, whereas on fibronectin (FN) or a substrate of molecules from heart cell-conditioned medium (HCM) a significant number of the cells died during early culture periods. Less than 70 and 50% of the number of neurons surviving on a laminin substrate were found on FN and HCM, respectively, after 3 days in culture. Although NGF did not affect neuronal survival, a small increase in neurite extension on these substrates was observed in the presence of NGF. Furthermore, although NGF did not prevent neuronal death after extended culture periods, this could be prevented by elevated extracellular potassium concentrations. Sympathetic neurons of E8 chick embryos however showed a strikingly different response to NGF compared with those of E7: whereas neuronal survival on laminin was not influenced by NGF, a significant effect of NGF on survival and on neurite extension was observed for E8 neurons on a HCM substrate. In contrast to cells from E7 and E8 embryos, the majority of neurons from E11 chick embryos required NGF for survival even on a laminin substrate as described previously (D. Edgar, R. Timpl, and H. Thoenen, 1984, EMBO J. 3, 1463-1468). These results demonstrate that while sympathetic neurons from E7 chick embryos do not depend on the soluble neurotrophic factor NGF for survival in vitro, they are dependent on molecules of the extracellular matrix. With increasing age, the survival requirements demonstrated in vitro change toward the classical pattern of NGF dependency. Low amounts of laminin-like immunoreactivity were shown to be present in sympathetic ganglia of E7 chick embryos which were then shown to increase as development proceeded. These data indicate that laminin may play a role in the survival and development of chick sympathetic neurons not only in vitro, but also in vivo.

Animals↗

PubMed1989

Neuronal laminin receptors.

The ability of the extracellular matrix protein laminin to regulate the survival and differentiation of neurons has led to the search for a neuronal laminin receptor. This article reviews the evidence for the existence of laminin receptors, and then goes on to discuss the difficulties in making the jump from a pharmacological demonstration of the receptor to its molecular identification.

Animals↗

PubMed1989

Developmental loss of functional laminin receptors on retinal ganglion cells is regulated by their target tissue, the optic tectum.

The ability of chick retinal ganglion cells (RGCs) to extend neurites on tissue culture substrata of the extra-cellular matrix protein laminin is lost during embryonic development. In order to establish the mechanism responsible for the loss of response, the number of high affinity (KD 10(-9) M) laminin receptors on both the cell bodies and neurites of RGCs were determined throughout this period by a ligand binding assay using radio-labelled laminin. It was found that the loss of response paralleled a decrease in receptor numbers on both the cell bodies and the neurites of the RGCs. Bilateral tectal ablation at embryonic day 6 resulted in the subsequent maintenance of laminin-stimulated neurite outgrowth, together with a partial inhibition of the loss of laminin receptors. Thus, the loss of response of the RGCs to laminin reflects a decrease in the numbers of laminin receptors on these neurons, and furthermore, this down-regulation is in turn dependent on innervation of the target tissue.

Animals↗

PubMed1988

Structural requirements for the stimulation of neurite outgrowth by two variants of laminin and their inhibition by antibodies.

Laminin derived from the Engelbreth-Holm-Swarm (EHS) tumor and a lamininlike molecule synthesized by RN22 Schwannoma cells both stimulate rapid neurite outgrowth, consistent with a common neurite-promoting site. However, antilaminin antisera can only inhibit the activity of the EHS laminin. The blocking antibodies in such sera are directed against the terminal heparin-binding domain of the laminin long arm (Edgar, D., R. Timpl, and H. Thoenen. 1984. EMBO [Eur. Mol. Biol. Organ.] J. 3: 1463-1468). These epitopes are demonstrated by immunoblotting to be part of the A chain and to be absent in RN22 laminin, showing (through metabolic labeling) that the cells synthesized little if any 440-kD A chain. This indicates that the antibody inhibition was probably due to steric hindrance, a common neurite-promoting site, apparently not being antigenic in native molecules. Antibodies raised against a 25-kD proteolytic fragment derived from the long arm of laminin were then used as probes to identify other potential neurite-promoting structures. Although these antibodies do not cross-react with native laminin, they recognized the B chains of denatured EHS and RN22 molecules on immunoblots. The antibodies also bound to the large proteolytic fragment, derived from the long arm of laminin that contains the neurite-promoting site, thus inhibiting its activity. Taken together, these results point to the localization of normally nonantigenic, defined, B chain sequences within or close to the neurite-promoting site of laminin.

Animals↗

PubMed1988

Avian corneal nerves: co-distribution with collagen type IV and acquisition of substance P immunoreactivity.

Within the avian cornea collagen type IV is preferentially and characteristically localized to the epithelial and endothelial basement membranes. In the present paper, we demonstrate that collagen type IV also is present within the corneal stroma coincident with the development and distribution of corneal nerves indicating that intra-stromal fibers are associated with Schwann cells or an equivalent cell type. We also demonstrate intra-stromal fibers of collagen type IV orthogonal to the epithelial basement membrane. These novel structures are most prominent on the tenth day of development and become progressively less distinct until they are no longer detectable on the eighteenth day of development. Substance P immunoreactivity is prominently expressed by nerves innervating the epithelium. The first substance P immunoreactive nerves are detected on the twelfth day of development, concomitant with the initiation of epithelial innervation and not the extension of nerves through the stroma. Such nerve fibers become more numerous with progressive development and demonstrate extensive association with both basal and superficial epithelial cells. Thus, the avian cornea is richly supplied with substance P primary afferents. The expression of substance P immunoreactivity correlates directly with the initiation of innervation of the corneal epithelium.

Animals↗

PubMed1987

The cellular interactions of laminin fragments. Cell adhesion correlates with two fragment-specific high affinity binding sites.

The molecular interactions of laminin with several tumor cell lines and skin fibroblasts were investigated by radioligand binding studies and cell attachment assays using laminin, the laminin-nidogen complex, and laminin fragments as substrates and also domain-specific antibodies as inhibitors of cell attachment. The majority of cells showed a dual binding pattern for fragments 1 and 8 which originate from short-arm or long-arm structures of laminin, respectively. Both of these fragments in solution bind to suspended cells with high affinity (KD = 1-10 nM), with the receptor numbers for each fragment depending on the cell type. Competition studies and independent variation of receptor numbers demonstrated that the cell-binding structures on each fragment are different, implicating the existence of two distinct cellular receptors for laminin. The ability of these fragments to act as substrates for cell adhesion correlated with the presence of high affinity binding sites on the cells. However, only antibodies to fragment 8 were able to block cell adhesion to laminin, despite the presence of binding sites for fragment 1. A few cells had very low numbers of high affinity receptors for either fragment 1 or 8. The latter cell type was used to demonstrate that complex formation between laminin and nidogen, which binds to fragment 1 structures, reduces the potential of laminin for cell binding.

Animals↗

PubMed1987

Distinct neurotrophic factors from skeletal muscle and the central nervous system interact synergistically to support the survival of cultured embryonic spinal motor neurons.

Motor neurons isolated from 6-day-old embryonic chick spinal cords require muscle extract for survival in culture; however, it was found, that some motor neurons, identified by retrograde labeling with rhodamine, will survive in mixed spinal cell cultures in the absence of the extract. The motor neuron survival-promoting activity produced by spinal cells is soluble and differs from the factor present in muscle extract, the two activities acting in a synergistic manner: the spinal cell activity potentiated that of muscle to decrease its ED50 by an order of magnitude, the motor neuronal survival (30%) seen in the presence of both factors being more than the sum of their individual activities. This synergism was shown to be restricted to the action of the spinal cell factor on motor neurons, no effect of the factor being noted with sympathetic neurons. As a series of defined growth and survival factors present in the central nervous system (nerve growth factor, brain-derived neurotrophic factor, acidic and basic fibroblast growth factors) had no effect on motor neuron survival, we conclude that the molecule responsible for the motor neuron survival-promoting activity of the spinal cells is a previously undefined factor.

Animals↗

PubMed1986

Muscle-derived factors that support survival and promote fiber outgrowth from embryonic chick spinal motor neurons in culture.

The purposes of the experiments reported is to provide an unambiguous demonstration that embryonic skeletal muscle contains factors that act directly on embryonic spinal motor neurons both to support their survival and to stimulate the outgrowth of neurites. Cells of lumbar and brachial ventral spinal cords from 6-day-old chick embryos were separated by centrifugation in a two-step metrizamide gradient, and a motor neuron enriched fraction was obtained. Motor neurons were identified by retrogradely labeling with rhodamine isothiocyanate, and were enriched fourfold in the motor neuron fraction relative to unfractionated cells. In culture, the isolated motor neurons died within 3-4 days unless they were supplemented with embryonic chick skeletal muscle extract. Two functionally distinct entities separable by ammonium sulfate precipitation were responsible for the effects of muscle extracts on motor neurons. The 0-25% ammonium sulfate precipitate contained molecules that alone had no effect on neuronal survival but when bound to polyornithine-coated culture substrata, stimulated neurite outgrowth and potentiated the survival activity present in muscle. Most of this activity was due to a laminin-like molecule being immunoprecipitated with antisera against laminin, and immunoblotting demonstrated the presence of both the A and B chains of laminin. A long-term survival activity resided in the 25-70% ammonium sulfate fraction, and its apparent total and specific activities were strongly dependent on the culture substrate. In contrast to the motor neurons, the cells from the other metrizamide fraction (including neuronal cells) could be kept in culture for a prolonged time without addition of exogenous factor(s).

Animals↗

PubMed1986

Laminin increases both levels and activity of tyrosine hydroxylase in calf adrenal chromaffin cells.

We have investigated the effects of substrate-bound laminin on levels of enzymes of the catecholamine biosynthetic pathway in primary cultures of calf adrenal chromaffin cells. Laminin increases the levels of the enzymes tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyl-transferase. This effect is selective, in that levels of other enzymes (lactate dehydrogenase, aromatic amino acid decarboxylase, and acetylcholinesterase) are not increased. The effect of laminin can be blocked by antibodies directed against a fragment of the heparin-binding domain of the molecule, whereas antibodies directed against other fragments do not block the increase in tyrosine hydroxylase. Thus the laminin domain involved in enzyme regulation in chromaffin cells is apparently the same as that previously implicated in laminin's interactions with neurons to potentiate survival and stimulate neurite outgrowth (Edgar, D., R. Timpl, and H. Thoenen, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1463-1468). The increase in chromaffin cell tyrosine hydroxylase levels is preceded by an activation of the enzyme in which the Vmax (but not the Km) is altered. The effects of laminin appear to be developmentally regulated, since neither activation nor increased levels of tyrosine hydroxylase occur in adult adrenal chromaffin cells exposed to laminin.

Animals↗

PubMed1986

Basement membrane proteins produced by Schwann cells and in neurofibromatosis.

Mouse Schwann cells and rat RN22 schwannoma cells cultured in the absence of neurons and fibroblasts produce typical basement membrane proteins. Heparan sulfate proteoglycan (low density form), nidogen, and protein BM-40 were identified by radioimmunoassays, immunoblotting, and by immunoprecipitation after metabolic labeling. The cells also produce a laminin-like protein that differs from authentic laminin by a reduced A chain content and lack of antigenic determinants located in the long arm of laminin. Laminin possessing A and B chains is, however, produced by PYS-2 teratocarcinoma cells grown under the same conditions. Laminin from Schwann cell culture medium promotes neurite outgrowth, and this activity could be immunoprecipitated but not blocked by various antibodies against authentic laminin. In addition, Schwann cell laminin is found complexed noncovalently with nidogen. Sulfate incorporation revealed the synthesis of proteoglycans and entactin. A similar set of proteins and in addition collagen IV could be demonstrated in neurofibroma tissue by immunohistology, and were localized to the laminae densae of the multilayered basement membranes around Schwann cells and capillaries. Laminin purified from 0.5 M NaCl neurofibroma tissue extracts possessed both A and B chains. Nidogen was identified in a partially degraded form.

Animals↗

PubMed1985

Neurotrophic factors.

In addition to nerve growth factor (NGF), many proteins present in soluble tissue extracts and in the extracellular matrix influence the survival and development of cultured neurons. The structure, synthesis, and mechanism of action of NGF as a neurotrophic factor are considered along with the experiments on the new putative trophic molecules.

Animals↗

PubMed1985

Senescence of a human lymphoblastoid clone producing anti-Rhesus(D).

The exploitation of Epstein-Barr virus transformation to generate human lymphoblastoid clones (LCL) producing antibody of predefined specificity has proved highly inefficient. Observations reported here on a cloned LCL producing anti-Rhesus(D) may provide an explanation for the low success rate. Over a few months this clone manifested a progressive loss of capacity to maintain growth in culture. Evidence consistent with terminal differentiation to a cell with the properties of a nonproliferating plasma cell was obtained. These late cells differed from those in the earlier actively cycling phase in that they were no longer able to respond to autostimulatory growth factors although they continued to produce them. This irreversible senescence leading to the death of the clone may be a common feature of virally transformed B cells and this would explain many of the difficulties encountered on this route to the production of human monoclonal antibodies.

Cell Survival↗

PubMed1985

Nerve growth factor changes the relative levels of neuropeptides in developing sensory and sympathetic ganglia of the chick embryo.

The effects of chronic nerve growth factor administration on the development of neuropeptides in the embryonic chick peripheral nervous system were quantitated by radioimmunoassays. Starting at embryonic Day 3.5, daily doses of 20 micrograms of nerve growth factor (NGF) increased the substance P content of lumbosacral spinal sensory ganglia at all ages studied (Days 10-14), while having no effect on substance P levels of thoracic sensory ganglia. In contrast, the contents of somatostatin were increased in both thoracic and lumbosacral ganglia, but only at comparatively late time points (Day 14). Nerve growth factor administration was also found to decrease the somatostatin contents of lumbosacral paravertebral sympathetic ganglia at early time points (Day 8) while increasing levels at later stages (Day 14), thus acting to accelerate the normally occurring developmental changes in level of this peptide. These changes were shown to be specific for somatostatin by demonstrating that NGF increased tyrosine hydroxylase levels in sympathetic neurons at Day 8, and had no effect on sympathetic vasoactive intestinal polypeptide levels at Day 14. It has been concluded that exogenous NGF does not simply act to increase or prolong the expression of neuron-specific phenotypes in the chick, but rather its action is time and location dependent to accelerate development.

Animals↗

PubMed1985

Nerve growth factors and molecules of the extracellular matrix in neuronal development.

The survival of developing neurons is epigenetically regulated by trophic factors. Only one such protein, the nerve growth factor (NGF) has been shown to act in vivo, where it supports the survival of neural-crest-derived sensory and sympathetic neurons. Recently, however, other proteins have been isolated and shown to support the survival of cultured neurons. Furthermore, in addition to the effects of soluble trophic factors, proteins of the extracellular matrix are also able to modulate neuronal survival. Analysis of the basal lamina protein, laminin, shows that when used as a culture substrate it stimulates neurite outgrowth and potentiates neuronal survival via a site associated with its heparin binding domain. On proteolytic cleavage of laminin, however, a cryptic site is unmasked that can also promote neuronal survival and neurite growth. The properties of this cryptic site indicate that it may be similar to that of the laminin-like molecule synthesized by Schwann cells, which although recognized by anti-laminin antibodies is not inhibited by them.

Animals↗

PubMed1984

The heparin-binding domain of laminin is responsible for its effects on neurite outgrowth and neuronal survival.

The survival of cultured chick sympathetic neurons and the outgrowth of neurites were stimulated by the basement membrane protein laminin coated onto polyornithine culture substrates. The survival-potentiating activity was dependent on the presence of nerve growth factor. Both effects of laminin could be completely inhibited by affinity-purified antibodies against laminin fragment 3, the product of a limited proteolysis that corresponds to the heparin-binding globular domain at the end of the long arm of the laminin molecule. Antibodies against other laminin fragments were inactive, including those against previously determined cell-binding domains. A large laminin fragment, E8, was produced by brief elastase digestion and shown to consist of fragment 3 and an adjacent rod-like structure. Although lacking the cell binding domains, fragment E8 potentiated both neuronal survival and neurite outgrowth, and these effects could be blocked by antibodies against fragment 3. Weak survival and neurite potentiating activity was also detected in another fragment corresponding to the short arms of laminin, but as these effects were not inhibited by any of the antibodies tested they probably arose de novo during proteolysis. The heparin-binding domain of laminin is therefore responsible for its effects on neurons.

Animals↗

PubMed1983

Both nerve growth factor and high K+ concentrations support the survival of chick embryo sympathetic neurons. Evidence for a common mechanism of action.

Neurons were dissociated from the sympathetic ganglia of embryonic chicks, and cultured in the absence of non-neuronal cells. Both nerve growth factor (NGF) and high concentrations of extracellular K+ supported neuronal survival, and these effects were independent of the presence of serum in the culture medium. Only 60% of the neurons survived in response to 35 mM K+, and survival was not increased when both NGF and K+ were present together. It was, however, possible to maintain essentially all the neurons in culture with either NGF or high K+ concentrations if the culture substrate had been pretreated with heart cell-conditioned medium (which did not itself support neuronal survival). These observations are consistent with a common mechanism of action of both K+ and NGF for the survival of cultured embryonic neurons.

Animals↗