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Biomedical subjects

T Ebendal

Publications and source records attributed to T Ebendal.

At least 91 records · Page 5Linked to original sources
PubMed1992

Neuritogenesis on collagen substrates. Involvement of integrin-like matrix receptors in retinal fibre outgrowth on collagen.

Extracellular matrix molecules such as laminin, fibronectin and collagen promote neurite outgrowth in vitro. We have investigated the capacity of hydrated gels of collagen types I-III and monomeric collagen types I-VI on plastic surfaces to support neuritogenesis. The attachment and survival of explants from the day 6 chick embryo were studied and neurite outgrowth measured as mean elongation rate and maximal neurite length. Collagen types I and III, both as three-dimensional gels or as native monomers supported neuritogenesis equal to or better than laminin. Collagen type V also supported neurite out-growth although less effectively. Collagen types II, IV and VI, as well as denatured collagens of all types tested, did not support outgrowth. The monoclonal anti-beta 1 integrin antibody (CSAT), as well as rabbit polyclonal antibodies directed to the integrin beta 1-chain, effectively inhibited neurite outgrowth on permissive collagenous substrata, indicating that collagen-binding integrins were involved in the neuritogenesis. These beta 1-integrins were independent of Arg-Gly-Asp (RGD) since neurite formation proceeded in the presence of synthetic RGD-containing peptides. Fluorescence immunohistochemistry revealed the presence of the integrin beta 1-chain on the outgrowing neurites. The results suggest a possible function of collagen and collagen-binding integrins in the development of the visual system.

Animals↗
PubMed1992

Effects of low-dose X-irradiation on mouse-brain aggregation cultures.

Biochemical and morphological differentiation in reaggregating mouse-brain cell cultures after low-dose radiation (0.5 Gy) in vitro was studied. Cells were irradiated on culture day 2, corresponding to embryonic day 15-16, and different glial and neuronal markers were followed through development to postnatal day 40. The shape and size of irradiated aggregates were more irregular and smaller compared with controls. Total amounts of DNA and protein were significantly lower in irradiated aggregates than in controls between days 8 and 20. After 30 days in culture activities of the glial markers glutamine synthetase (GS) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) were lower in X-irradiated aggregates than in controls. However, after 40 days the CNP activity in irradiated aggregates increased to levels above those of the controls. Irradiated and control aggregates did not differ significantly in neuronal marker enzyme activities, i.e. choline acetyltransferase (ChAT), acetylcholine esterase (AChE) and glutamic acid decarboxylase (GAD) measured on a per mg protein basis. On days 20 and 30 the amount of nerve growth factor (NGF) was two-fold higher in irradiated aggregates compared with non-irradiated ones, suggesting that, after irradiation, surviving cells in culture were induced to produce more NGF. After 40 days the amount of NGF in irradiated aggregates had decreased to the level found in the control aggregates.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1992

No re-expression of high-affinity nerve growth factor binding sites in spinal motor neurons in amyotrophic lateral sclerosis.

Autoradiographic studies of high-affinity nerve growth factor binding sites in the cervical human spinal cord revealed a high density of binding sites in the dorsal horn and in the tract of Lissauer, which is in agreement with investigations in nonprimates. Very low or no binding was found in the motor neuron areas of controls as well as in amyotrophic lateral sclerosis cases, which indicates that the degeneration of motor neurons in this disease is not accompanied by re-expression of high-affinity nerve growth factor receptors.

Aged↗
PubMed1991

Methylmercury induced alterations in the nerve growth factor level in the developing brain.

Pre- and early postnatal stages in the development of the central nervous system (CNS) are very sensitive to the toxic effects of methylmercury. The influence of methylmercury on the level of nerve growth factor (NGF) during the development of CNS was studied. Sprague-Dawley rats were exposed indirectly throughout the fetal and suckling periods until weaning on postnatal day 25 (P 25) via their dams given methylmercury in the diet (3.9 mg/kg diet). In addition, after weaning offsprings were exposed directly to methylmercury via the diet until postnatal day 50 (P 50). The level of NGF was analyzed in cortical areas and in the septum with a sensitive enzyme immunoassay. The pups exposed to MeHg exhibited a 50% elevation in the level of NGF in the hippocampus on P 25 and P 50 compared to control animals. Concomitantly, the level of NGF decreased by 30% in the septum on P 25 and P 50, suggesting that the retrograde transport of NGF from hippocampus to septum could be affected by the exposure of methylmercury. The exact mechanism by which the low level of mercury is affecting the NGF concentration in the developing brain is yet unknown. The increase of NGF in the hippocampus and the decrease of NGF measured in the septum could reflect altered conditions for neurotrophic support in these areas of the brain as a result of the exposure to heavy metal. Thus, this finding might indicate a connection between exposure of heavy metals and neurodegeneration, such as that found in the basal forebrain in Alzheimer's disease.

Animals↗
PubMed1991

Intraputaminal infusion of nerve growth factor to support adrenal medullary autografts in Parkinson's disease. One-year follow-up of first clinical trial.

Experimental studies in rodents show that beta-nerve growth factor can increase the survival, neurite outgrowth, and functional effect of grafts of adrenal chromaffin cells to the basal ganglia. We, therefore, have begun to investigate whether treatment with nerve growth factor might also increase the functional effect of autografts of adrenal medullary tissue in patients with Parkinson's disease. Previous studies have shown that stereotactic implantation of adrenal tissue pieces produces a transient functional improvement that lasts for a few months. This report describes a trial of grafting of adrenal chromaffin tissue into the putamen, supported by infusion of nerve growth factor. The patient is a 63-year-old woman with a 19-year history of Parkinson's disease, now complicated by on-off phenomena and drug-induced hyperkinesia, despite optimized medical management. The left adrenal gland was removed, and the medulla was dissected into 1- to 2-mm3 pieces in a solution containing nerve growth factor purified from mouse submandibular gland. Pieces were implanted in six tracts 3 to 4 mm from a previously placed cannula in the left putamen. Through the cannula, nerve growth factor was infused for 23 days for a total dose of 3.3 mg. Clinical assessment consisted of global ratings for rigidity and/or hypokinesia and for drug-induced hyperkinesia. Measures of gait and fine-motor control were also made. The motor readiness potential and auditory evoked potentials were recorded.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗
PubMed1991

Chimeric molecules with multiple neurotrophic activities reveal structural elements determining the specificities of NGF and BDNF.

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are two members of a family of neurotrophic factors which show both overlapping and distinct neurotrophic activities. Using site-directed mutagenesis, chimeric molecules were constructed where different combinations of sequences from BDNF replaced the corresponding sequences in NGF. The resulting molecules were transiently expressed in COS cells and conditioned media containing the chimeric proteins were assayed for biological activity in explanted chick sympathetic, spinal and nodose ganglia. Our results show that the biological specificities of the two proteins are obtained by specific combinations of a set of sequences that differ between the two molecules. Some of these combinations allowed us to engineer molecules which display multiple neurotrophic activities recruited from both the NGF and BDNF proteins.

Amino Acid Sequence↗
PubMed1991

Messenger RNAs for trk and the low-affinity NGF receptor in rat basal forebrain.

Nerve growth factor (NGF) acts by binding to specific sets of neurons. Low-affinity binding (Kd of 10(-9) M) is mediated via a 75 kDa glycoprotein (LNGFR). Recently, a 140 kDa tyrosine protein kinase encoded by the proto-oncogene trk has been found to bind NGF with high affinity (Kd of 10(-11) M) and to evoke neurotrophic responses. In addition, the related trkB protein and the LNGFR have been shown to bind brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) with an affinity of Kd 10(-9) M. We demonstrate the distribution of mRNA encoding the neurotrophin-binding proteins in the forebrain of the 21-day-old Sprague-Dawley rat by in-situ hybridization. Expression of trk and LNGFR mRNA showed co-localization and was restricted to the medial septal nucleus and the nucleus of Broca's diagonal band, suggesting a receptor function in these cells for both proteins encoded. In contrast, expression of trkB was widely spread in many areas. Thus trkB protein might serve general functions in the forebrain.

Aging↗
PubMed1991

Biological and immunological properties of recombinant human, rat, and chicken nerve growth factors: a comparative study.

Biological and immunological properties of recombinant human, rat, and chicken nerve growth factors (NGFs) were studied and compared. Recombinant NGF proteins were produced in a transient expression system using COS cells and levels of secreted NGF protein were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of conditioned media from in vivo [35S]cysteine-labeled cell cultures. Antigenic differences among the three NGFs were studied by immunoblotting and immunoprecipitation of secreted cell products using a rabbit polyclonal antiserum against purified mouse NGF, and by a two-site enzyme immunoassay (EIA) with a monoclonal antibody against mouse NGF. Although all three NGFs were recognized equally well in the immunoblotting, only one-third of the chicken NGF protein could be detected by immunoprecipitation or by the EIA as compared to the rat and human NGFs. Thus, changes in the three-dimensional structure of the NGF molecule are most likely responsible for the antigenic differences between avian and mammalian NGFs. The three NGF proteins were also compared in their ability to displace 125I-mouse NGF from low-affinity NGF receptors on rat pheochromocytoma PC12 cells. Similar displacement curves and values were obtained for each NGF protein, indicating that structural differences among these molecules do not affect low-affinity binding to NGF receptors. Biological activities were studied by the ability of the conditioned media to promote neurite outgrowth from explants of E9 chick sympathetic ganglia and from PC12 cells. Although the rat system showed a slight preference for the homologous molecule, the morphological changes, dose-response curves, and maximal stimulation values obtained with the different NGFs were practically indistinguishable in the chicken bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Gland Neoplasms↗
PubMed1991

Thyrocyte migration and histiotypic follicle regeneration are promoted by epidermal growth factor in primary culture of thyroid follicles in collagen gel.

The effect of epidermal growth factor (EGF) on the morphology of porcine thyroid follicles cultured in collagen gel was studied by light and electron microscopy. Treatment with EGF (10 ng/ml) for 1-4 days induced a progressive migration of thyrocytes radiating from the mother follicles into the collagen lattice. Migrating cells were often connected with junctional complexes, thus forming small follicles containing microlumina, in which the limiting portion of the plasma membrane expressed microvilli. Autoradiography of [3H]thymidine incorporation in EGF-treated cultures showed that more than 80% of nuclei in mother follicles and migrating cells were labeled after 4 days. TSH (2 mU/ml) given simultaneously with EGF did not influence the effect of EGF on multiplication, migration, and formation of microfollicles. TSH given for 12 h after 4 days of exposure to EGF was able to induce luminal dilation of mother follicles, but caused no change in the appearance of microlumina. It is concluded that thyrocytes in collagen gel retain epithelial characteristics during prolonged stimulation with EGF, in spite of a migrating response. The whole sequence of events, starting with the intact follicle, i.e. multiplication, migration of thyrocytes, and formation of new follicles, could, thus, be visualized to occur in response to a single mitogen, EGF. EGF might be involved in the generation of new follicles in the intact gland.

Animals↗
PubMed1990

Environmental influence on behaviour and nerve growth factor in the brain.

The influence of the environment on the endogenous levels of nerve growth factor (NGF) in the cortex, hippocampus and septum was examined in adult (82 days old) and juvenile (51 days old) rats. Animals were reared/housed for 30 days in an enriched, standard or isolated environment prior to analysis. In addition, another group of rats were given behavioural tests (4 days) after differential rearing/housing before measurements of NGF. We found complex variations in the level of NGF both in juvenile and adult hippocampus after differential environmental rearing/housing. Rearing/housing in an enriched environment improved performance in the Morris maze and decreased spontaneous motor activity. Exposure to behavioural tests caused alterations in adult hippocampus and septum NGF levels. The results show that testing in a novel environment causes small but significant changes in the hippocampal and septal NGF levels depending upon the environmental history of the animal. In view of the purported involvement of the septohippocampal pathway and NGF in the pathophysiology of Alzheimer's disease, our finding suggests that lack of adequate environmental stimulation might be of importance in age-related behavioural and neurochemical deficits.

Analysis of Variance↗
PubMed1990

[Nerve growth factor as a therapeutic agent: transplantation of brain tissue as a possible treatment of neurodegenerative disorders].

An account of experiments focused on the treatment of neurodegenerative disease by means of cell grafts, in which genetically modified cells that release abundant nerve growth factor are described, and their transplantation to the rat brain reported. The cells can survive in the rat brain and exert potent effects on cholinergic neurons both in the host cortex, basal forebrain and striatum, and in tissue grafts.

Animals↗
PubMed1990

Structure-function studies of nerve growth factor: functional importance of highly conserved amino acid residues.

Selected amino acid residues in chicken nerve growth factor (NGF) were replaced by site-directed mutagenesis. Mutated NGF sequences were transiently expressed in COS cells and the yield of NGF protein in conditioned medium was quantified by Western blotting. Binding of each mutant to NGF receptors on PC12 cells was evaluated in a competition assay. The biological activity was determined by measuring stimulation of neurite outgrowth from chick sympathetic ganglia. The residues homologous to the proposed receptor binding site of insulin (Ser18, Met19, Val21, Asp23) were substituted by Ala. Replacement of Ser18, Met19 and Asp23 did not affect NGF activity. Modification of Val21 notably reduced both receptor binding and biological activity, suggesting that this residue is important to retain a fully active NGF. The highly conserved Tyr51 and Arg99 were converted into Phe and Lys respectively, without changing the biological properties of the molecule. However, binding and biological activity were greatly impaired after the simultaneous replacement of both Arg99 and Arg102 by Gly. The three conserved Trp residues at positions 20, 75 and 98 were substituted by Phe. The Trp mutated proteins retained 15-60% of receptor binding and 40-80% of biological activity, indicating that the Trp residues are not essential for NGF activity. However, replacement of Trp20 significantly reduced the amount of NGF in the medium, suggesting that this residue may be important for protein stability.

Amino Acid Sequence↗
PubMed1990

Rescue of basal forebrain cholinergic neurons after implantation of genetically modified cells producing recombinant NGF.

Mouse 3T3 fibroblasts were genetically modified by transfection with a mammalian expression vector containing the rat beta-nerve growth factor (NGF) gene. The transfected cell line, designated 3E, contains several hundred copies of the rat NGF gene and secretes high levels of biologically active NGF. Pieces of collagen gel containing the NGF-secreting 3E cells were grafted to the brains of unilaterally fimbria-fornix-lesioned rats. Grafts of the genetically modified NGF-producing cells rescued axotomized basal forebrain cholinergic neurons and significantly reduced cholinergic cell death in the medial septum as compared with rats treated with grafts of the parental 3T3 cells. Grafted fibroblast cells were detected, and rescue effects were noted up to 6 weeks after grafting. Local effects of NGF secreted by grafted cells were also seen at the gel-brain border in the form of sprouting acetylcholinesterase immunoreactive host cortical fibers. We suggest that implantation of genetically modified cells producing NGF may have therapeutic applications in rescuing damaged central cholinergic neurons in senile dementia of the Alzheimer type as well as in providing trophic support for chromaffin tissue grafts in Parkinson's disease.

Acetylcholinesterase↗
PubMed1990

Regulation of beta-nerve growth factor expression by inflammatory mediators in hippocampal cultures.

Substances which regulate expression of nerve growth factor (NGF) were examined in embryonic rat hippocampal cultures containing both neurons and glial cells. Both cell types expressed NGF mRNA when cultivated in vitro. Lipopolysaccharide, an activator of macrophages, elicited a significant increase in NGF mRNA. Interleukin-1 beta evoked a similar increase in NGF mRNA which was accompanied by a rise in NGF protein. The Il-1-induced increase was partially blocked by indomethacin, suggesting that prostaglandins might mediate this effect. Treatment of the cultures directly with prostaglandin E2 resulted in elevated levels of both NGF mRNA and protein. Thus, agents which promote inflammatory activity appear to increase NGF expression. Moreover, a suppressor of inflammation, dexamethasone, decreased NGF expression. Our observations indicate that a variety of immunomodulators regulate NGF expression in the hippocampus.

Animals↗
PubMed1990

Recombinant human beta-nerve growth factor (NGF): biological activity and properties in an enzyme immunoassay.

Nerve growth factor (NGF) supports sympathetic and sensory neurons in the peripheral nervous system and also functions in the development and maintenance of cholinergic neurons in the basal forebrain. NGF distribution can be studied in the brain of the rat and mouse with the use of a sensitive two-site enzyme immunoassay (EIA) for mouse NGF. It would be of interest to measure the NGF protein also in the human brain, especially against the background that the cholinergic neurons are severely deteriorated in senile dementia of the Alzheimer type. The limited immunological cross-reactivity between NGFs from different species has previously hampered attempts to determine levels of the human NGF. We have now examined the biological activity and immunological properties of human recombinant NGF protein in medium conditioned by COS cells transfected with the human NGF gene. The human NGF behaved similar to mouse NGF in a sympathetic ganglion bioassay. The monoclonal antibody 27/21 to mouse NGF was shown to effectively block the activity of both the human recombinant NGF and mouse native NGF. A two-site EIA using monoclonal antibody 27/21 was optimized. Under the conditions used, the EIA detected the human recombinant NGF with the same sensitivity (1 pg/ml) as shown for the mouse NGF. It should now be possible to test this EIA also on homogenized tissue to examine human NGF in brain samples from Alzheimer patients and age-matched controls.

Amino Acid Sequence↗