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Biomedical subjects

D Edgar

Publications and source records attributed to D Edgar.

At least 37 records · Page 2Linked to original sources
PubMed1998

The targeted deletion of the LAMC1 gene.

Laminin is required for the production of a basement membrane in both the early embryo and in the embryoid body. The absence of the gamma 1 chain leads to different processing of the other subunits in the trimer and prevents the production of a polymerizing laminin molecule. Collagen IV and perlecan are not able to compensate for the loss of the laminin molecule, and nidogen, which would normally bind to the gamma 1 chain, is almost completely lost from the embryoid body. In the embryo the failure of the basement membrane results in embryonic lethality at embryonic day 5.5, a time when the primitive endoderm is differentiating to visceral and parietal endoderm, and the latter is migrating out of the inner cell mass over the trophectoderm. It is likely that failure in one or both of these events leads to the death of the embryo. The absence of the trophectodermal basement membrane normally present in the blastocyst appears not to be necessary for the formation of the blastocystic cavity, which depends on the formation of a polarized epithelium. Hence the first epithelium produced in the developing embryo does so independently of a basement membrane.

Animals↗
PubMed1997

Vitronectin is expressed in the ventral region of the neural tube and promotes the differentiation of motor neurons.

The extracellular matrix protein vitronectin and its mRNA are present in the embryonic chick notochord, floor plate and in the ventral neural tube at the time position of motor neuron generation. When added to cultures of neural tube explants of developmental stage 9, vitronectin promotes the generation of motor neurons in the absence of either notochord or exogenously added Sonic hedgehog. Conversely, the neutralisation of endogenous vitronectin with antibodies inhibits over 90% motor neuron differentiation in co-cultured neural tube/notochord explants, neural tube explants cultured in the presence of Sonic hedgehog, and in committed (stage 13) neural tube explants. Furthermore, treatment of embryos with anti-vitronectin antibodies results in a substantial and specific reduction in the number of motor neurons generated in vivo. These results demonstrate that vitronectin stimulates the differentiation of motor neurons in vitro and in vivo. Since the treatment of stage 9 neural tube explants with Sonic hedgehog resulted in induction of vitronectin mRNA expression before the expression of floor plate markers, we conclude that vitronectin may act either as a downstream effector in the signalling cascade induced by Sonic hedgehog, or as a synergistic factor that increases Shh-induced motor neuron differentiation.

Amino Acid Sequence↗
PubMed1996

Insulin-like growth factor-I stimulates neurogenesis in chick retina by regulating expression of the alpha 6 integrin subunit.

Insulin-like growth factor I (IGF-I) strongly stimulates the generation of differentiated neurons in cultures of neuroepithelial cells of the embryonic chick neural retina in the presence of a laminin-1 tissue culture substrate. Treatment of cultured neuroepithelial cells with IGF-I rapidly up-regulated the mRNA coding for the alpha 6 integrin subunit whereas specific reduction of alpha 6 subunit levels by treatment with an alpha 6 integrin antisense oligonucleotide resulted in reduced neuronal differentiation in vitro. Although IGF-I immunoreactivity is seen throughout the neural retina, expression of IGF-I mRNA is confined to the pigment epithelium during the period of neurogenesis in vivo. Neutralization of the endogenous IGF-I with a blocking antibody down-regulated levels of alpha 6 integrin mRNA and reduced the production of differentiated retinal neurons in vivo. These data indicate a role for IGF-I in the generation of retinal neurons mediated by the interaction of laminin with its alpha 6 integrin subunit-containing receptor.

Animals↗
PubMed1995

Reduced laminin immunoreactivity in the blood vessel wall of ageing rats correlates with reduced innervation in vivo and following transplantation.

Changes in extracellular matrix composition and/or organization, and in particular in the ratio of axonal growth-promoting components such as laminin to growth-inhibiting molecules, could contribute to the degenerative changes observed in the innervation of some peripheral tissues in old age. We have investigated this issue by evaluating laminin content or accessibility at various locations on blood vessels where we had previously studied age-related alterations in innervation density. We have employed a morphological approach, measuring laminin immunoreactivity by a densitometric application of confocal microscopy, because more conventional biochemical techniques would have been unable to distinguish specific, localized changes in laminin at sites accessible to nerves from heterogeneous changes in other areas of the vessel wall, such as the endothelial basal lamina. We found that in 24-month-old rats laminin immunoreactivity is decreased by 50% at the medial-adventitial border in association with the outer layer of smooth muscle cells, where a parallel decrease is observed in innervation density. Axonal terminals were shown to have access to laminin in this region of the blood vessel wall by double staining with laminin and a general neuronal marker. Changes in laminin immunoreactivity were region-specific on the same blood vessel, thus excluding the possibility of a generalized decrease in immunoreactivity in old age. For example, in the basilar artery intensity of laminin immunoreactivity decreased in old age at the medial-adventitial border, but showed no change in endothelial cell basal lamina and in the adventitia. Moreover, we performed in oculo transplants of blood vessels displaying differences in laminin immunoreactivity and found that the density of innervation correlated with the intensity of laminin staining, thus lending further support to the hypothesis that laminin might play a role in nerve fibre atrophy in old age.

Aging↗
PubMed1995

Abnormal expression and distribution of nidogen in Hirschsprung's disease.

Hirschsprung's disease (congenital colonic aganglionosis) is associated with abnormalities in the distributions and amounts of basement membrane and other extracellular matrix components in the human gut. The authors have investigated the possible significance of nidogen in Hirschsprung's disease, because this glycoprotein is necessary for the formation of ternary complexes with the other basement membrane components, laminin and collagen type IV, and thus may contribute the pathology of the disease. Increased nidogen immunoreactivity in the smooth muscle basement membranes and muscularis mucosae of the distal aganglionic zone in Hirschsprung's bowel was observed, the nidogen immunoreactivity demonstrating that the thickness of the muscularis mucosae was increased in this region. However, steady-state nidogen mRNA levels were found to be significantly lower in both proximal and distal Hirschsprung's bowel (relative to controls). In contrast, no significant differences were observed in the steady-state levels of the mRNAs coding for laminin subunits. These results indicate that although abnormalities in the amount or distribution of nidogen may contribute to the abnormalities seen in the extracellular matrix in Hirschsprung's disease, the levels of expression of the genes coding for either nidogen or laminin are unlikely to be primarily responsible for the lesions.

Basement Membrane↗
PubMed1995

Regulated expression of a novel laminin beta subunit during the development of the chick embryo.

In order to define specific laminin variants implicated in organogenesis, we have undertaken a systematic search to detect and characterize novel laminin subunits, the expression of which is both developmentally regulated and tissue-specific. cDNA prepared from embryonic chick tissues was amplified by the polymerase chain reaction (PCR) with degenerate primers based on conserved sequences in domains V and VI of members of the laminin beta subunit family. Restriction mapping, cloning and sequencing of PCR products demonstrated a novel cDNA, the derived protein sequence of which displayed greatest homology with laminin beta subunits. However, the degree of amino acid divergence, comparison of sequence motifs and pattern of expression indicates that the cloned cDNA does not code for the avian orthologue of a previously characterized laminin beta subunit. Northern blot analysis showed that the expression of the 6-kb mRNA coding for the novel subunit was restricted to the skin. The mRNA was not detectable before day 5 of chick embryonic development, after which in situ hybridization showed expression only in surface ectoderm cells and subsequently in the epidermis. The developmentally regulated ectodermal expression of the novel beta subunit, prior to condensation of mesenchymal cells to form the dermis, is consistent with a specific role for this laminin isoform in the development and maintenance of the skin.

Amino Acid Sequence↗
PubMed1994

The extracellular matrix components, tenascin and fibronectin, in Hirschsprung's disease: an immunohistochemical study.

Previous in vitro studies have suggested that successful neural crest cell migration in the developing gut could be influenced by the composition of the extracellular matrix components, tenascin and fibronectin. The authors aimed to gain insight into the pathogenesis of Hirschsprung's disease (HD) by studying the distribution of tenascin and fibronectin in bowel specimens of patients with HD. Immunohistochemical examination was performed in specimens from 10 HD patients (8 aganglionic, 5 transitional, and 10 normoganglionic zones) and 18 age- and site-matched controls undergoing other types of gastrointestinal surgery. The distribution of tenascin was restricted to the basement membranes of the smooth muscle and vasculature, and in the basement membranes surrounding neuronal ganglia in all the controls and in 10 proximal normoganglionic HD specimens. More intense tenascin immunofluorescence was observed in the smooth muscle basement membranes of the muscularis externa of eight aganglionic and five transitional zones of HD. Wide-spread distribution of fibronectin was found in all the basement membranes as well as in the lamina propria and submucosa of all control and 10 normoganglionic HD sections. However, more intense immunofluorescence with fibronectin was observed in all the layers of eight aganglionic and five transitional zones of HD specimens. The present findings show that the mesenchymal and basement membrane extracellular matrix constitution is abnormal in the affected bowel of HD. Although a causal relationship has not been demonstrated, corroborative evidence from earlier animal experiments in other studies suggests that the extracellular matrix abnormalities may contribute to the pathogenesis of HD.

Basement Membrane↗
PubMed1993

Developmental regulation of basement membrane deposition.

The expression and localisation of the basement membrane glycoprotein laminin in tissues are two factors that determine to what extent it can influence cell behaviour during development. In order to begin to unravel the potential functions of laminin in vivo, the mechanisms regulating its expression, structure and stabilization in the basement membranes of developing tissues are discussed.

Animals↗
PubMed1992

Instrumental comparison for the determination of cadmium and lead in calcium supplements and other calcium-rich matrices.

Three brands of Ca supplement, a laboratory-reagent grade CaCO3 and a certified reference material (International Atomic Energy Agency H-5 Animal Bone) wee analysed for Cd and Pb by four different analytical techniques, viz., anodic stripping voltammetry inductively coupled plasma mass spectrometry, flame atomic absorption spectrometry and electrothermal atomic absorption spectrometry. The Pb levels measured by the four techniques in the bone powder were within the certified Pb level in this certified reference material. Similarly, no significant differences [p less than 0.05; analysis of variance (ANOVA)] were observed in samples with Pb concentrations greater than 1 microgram g-1. However, the Pb levels in the laboratory-reagent grade CaCO3 obtained by flame atomic absorption spectrometry (0.79 micrograms g-1) averaged about three times higher than those measured by the other three techniques (i.e., 0.25 micrograms g-1). Although no significant differences (p less than 0.05; ANOVA) in Cd levels were observed within any of the samples (intra-sample variability), the Cd concentration measured in the different Ca supplements (inter-sample variability) varied by three orders of magnitude (ranging from 0.07 to 3.59 micrograms g-1).

Analysis of Variance↗
PubMed1992

Does catheter immobilization reduce exit-site infections in CAPD patients?

This study was undertaken to ascertain the effectiveness of an immobilization device in reducing exit site infections (ESI) in CAPD patients, and whether immuno-suppressive therapy, diabetes, disconnect system and Staphylococcus aureus nasal carriage had any bearing on the incidence of ESI. Sixty-six patients having a Tenckhoff catheter placement were randomly allocated into one of three groups; immobilizer, tape and non-immobilized group. The groups were monitored for the incidence of ESI over a total period of 347 patient months. The results show no significant difference in infection rates between the three groups, nor do the factors mentioned have any bearing on ESI rate. Whilst immobilization is important, the ineffectiveness of this device was probably related to its design problem.

Bacterial Infections↗
PubMed1992

Abnormalities in the distribution of laminin and collagen type IV in Hirschsprung's disease.

In vitro neurite outgrowth and neuronal survival are promoted by laminin, and neuronal migration is promoted by collagen type IV. This led to the hypothesis that Hirschsprung's disease (HD) could be the result of an abnormal extracellular matrix microenvironment in the affected bowel during embryogenesis. Using indirect immunohistochemistry, we studied the distribution of laminin and collagen type IV in the bowel specimens of eight HD patients (four neonates and four infants) and 16 age- and site-matched controls from non-HD patients. In eight HD specimens, the tissue studied was from the aganglionic, transitional, and proximal normoganglionic zones. Uniform distribution of laminin and collagen type IV was observed in the basement membranes of all control specimens. The semiquantitative abnormality in the distribution of these proteins in HD occurred as follows: immunoreactivities were more intense in the inner circular layer, diminishing with a gradient in the outer muscularis externa in six of six aganglionic, four of five transitional, and three of eight normoganglionic sections. The qualitative abnormality of these proteins in HD was speckled immunofluorescence outside the basement membranes of the muscularis externa in all three zones of neonatal specimens only. These findings support the microenvironment hypothesis of the pathogenesis of HD.

Basement Membrane↗
PubMed1991

The expression and distribution of laminin in the developing nervous system.

The extracellular matrix glycoprotein laminin exerts profound effects on the survival and differentiation of neurons in vitro. Although principally confined to the basement membranes of the adult extracellular matrix, during development laminin immunoreactivity may be found both within the interstitial extracellular matrix and on the membranes of neural cells, in which location it may be expected to affect their development in vivo. To investigate the reasons for the occurrence of laminin outside basement membranes, the expression of genes coding for laminin subunits has been analysed in mouse sciatic nerves at different postnatal ages. The results show that the expression of genes coding for laminin subunits decreases from high levels at birth, very low steady state levels of the individual mRNAs being found in the adult. Thus, the predicted high rate of laminin synthesis during development might exceed the rate at which it may be incorporated into basement membranes. The effective change in distribution of laminin into the basement membrane is therefore likely to be simply a consequence of the down-regulation of gene expression, rather than being caused, for example, by the differential expression of laminin variants.

Animals↗
PubMed1990

Cell adhesion, spreading and neurite stimulation by laminin fragment E8 depends on maintenance of secondary and tertiary structure in its rod and globular domain.

The cell adhesion, spreading and neurite-promoting properties of mouse tumor laminin fragment E8, which contains major site(s) responsible for laminin-cell interactions, were probed by proteolytic degradation, denaturation, synthetic peptides and antibody inhibition. Removal of more than half of the N-terminal portion contributing to the rod-like domain did not effect cell attachment or spreading although neurite-promoting activity was reduced. More extensive degradation of the rod or of the globular domains of E8, or separation of the globule from the rod, also resulted in loss of cell spreading activity although weak attachment was found to an A chain subfragment comprising the globular domain and a short piece of the rod. Exposure of E8 to increasing concentrations of dissociating agents produce an apparently reversible denaturation but an irreversible loss of both attachment and neurite-promoting activities, as did reduction and alkylation of disulfide bonds in the globular domain. Although cell adhesion and spreading were blocked by antibodies to an alpha 6 integrin subunit, neurite outgrowth was unaffected, indicating two distinct receptors for these two activities. Furthermore, a synthetic peptide, the sequence of which is found in the vicinity of adhesion and neurite-promoting sites and previously implicated in neurite growth and cell attachment activities, was found to be inactive. These results indicate that the major cell attachment and neurite-promoting sites of laminin are distinct although both require the native conformation of parts of the rod and the terminal globular domain of the long arm of laminin.

Amino Acid Sequence↗
PubMed1990

Immunoelectron microscopic localization of laminin in normal and regenerating mouse sciatic nerve.

Laminin, the major non-collagenous protein of basement membranes, has been shown to be a potent stimulator of neurite outgrowth, to potentiate neuronal survival and to stimulate Schwann cell division in vitro. The aim of the present study was to determine the ultrastructural localization of laminin in the mouse sciatic nerve during development and regeneration in order to elucidate whether laminin might also evoke similar effects in vivo. For this purpose polyclonal antibodies against laminin were used for pre-embedding electron microscopic immunocytochemistry of tissue sections from mouse sciatic nerves. In the adult, although laminin immunoreactivity was found to be predominantly associated with basement membranes as expected, the surface membranes of Schwann cells also displayed weak labelling. This distribution pattern was similar in developing sciatic nerves with the exception that laminin immunoreactivity was generally higher and also found to be present on interstitial collagen fibres. One week following sciatic nerve transection, strong laminin immunoreactivity was seen on regenerating axons growing along laminin-positive basement membrane tubes in the distal stump of the transected nerve. Our results demonstrate that laminin immunoreactivity is not restricted to basement membranes of the mouse sciatic nerve, but is also found in direct contact with adult, developing and regenerating axons as well as on the surface of Schwann cells. The finding of laminin immunoreactivity on extracellular matrix components, axons and Schwann cell membranes under conditions of growth and regeneration makes it more likely that axons are able to interact with laminin not only in vitro but also in vivo.

Aging↗