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T Ebendal

Publications and source records attributed to T Ebendal.

At least 109 records · Page 6Linked to original sources
PubMed1990

Molecular cloning and neurotrophic activities of a protein with structural similarities to nerve growth factor: developmental and topographical expression in the brain.

We have used a pool of degenerate oligonucleotides representing all possible codons in regions of homology between brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) to prime rat hippocampal cDNAs in the polymerase chain reaction. The amplified DNA included a product with significant similarity to NGF and BDNF, which was used to isolate a 1020-nucleotide-long cDNA from a rat hippocampal library. From the nucleotide sequence, a 282-amino-acid-long protein with approximately 45% amino acid similarity to both pig BDNF and rat NGF was deduced. In the adult brain, the mRNA for this protein was predominantly expressed in hippocampus, where it was confined to a subset of pyramidal and granular neurons. The developmental expression in brain showed a clear peak shortly after birth, 1 and 2 weeks earlier than maximal expression of BDNF and NGF, respectively. It was also expressed in several peripheral tissues with the highest level in kidney. The protein, transiently expressed in COS cells, was tested on chicken embryonic neurons and readily stimulated fiber outgrowth from explanted Remak's ganglion and, to a lesser extent, the nodose ganglion. A weak, but consistent, fiber outgrowth response was also seen in the ciliary ganglion and in paravertebral sympathetic ganglia. Moreover, the protein displaced binding of NGF to its receptor, suggesting that it can interact with the NGF receptor. Thus, this factor, although structurally and functionally related to NGF and BDNF, has unique biological activities and represents a member of a family of neurotrophic factors that may cooperate to support the development and maintenance of the vertebrate nervous system.

Aging↗
PubMed1990

Expression of nerve growth factor receptor mRNA during early development of the chicken embryo: emphasis on cranial ganglia.

In situ hybridization with beta-nerve growth factor receptor (NGF-R) oligonucleotide probes was used to study NGF-R mRNA expression in early chicken embryos. Sections through the region of the visceral arches showed high levels of NGF-R mRNA in mesenchyme of the visceral arches, neural tube and myotomes. Labelling was also seen over E3 primordium of the trigeminal ganglion (V) and in the placodal thickening of the petrosal (IX) and nodose (X) ganglionic primordia. In the E5 embryo, all cranial sensory ganglia (V, VII, VIII, IX, X) expressed NGF-R mRNA although at varying levels with higher levels in the ganglia of the Vth, IXth and Xth cranial nerves than in ganglia of the VIIth and the VIIIth nerves. Within ganglia of the Vth, IXth and Xth cranial nerves, levels of NGF-R mRNA were higher in regions containing placode-derived neurons, than in regions with neural-crest-derived neurons. The placode-derived nodose ganglion (X) expressed NGF-R mRNA at all stages of development. In the E15 embryo and later in development, two thirds of the large neuron-like cells expressed high levels of NGF-R mRNA. Our results show that expression of NGF-R mRNA, in peripheral neurons, is not restricted to cells of neural crest origin. We also show a transient expression of NGF-R mRNA early in development in a wide range of non-neuronal differentiating cells. The high level of NGF-R mRNA in early differentiating tissues suggest that the NGF-R plays a wider role during development than previously anticipated.

Animals↗
PubMed1989

Characterization of antibodies to synthetic nerve growth factor (NGF) and proNGF peptides.

Sequence data for the mature nerve growth factor (NGF) protein and its precursor are available from molecular cloning of the NGF gene in several species, including mice, humans, rats, and chickens. Hydrophilicity analysis of the predicted rat and chicken prepro-NGF was carried out to locate putative antigenic determinants. Eight peptides were selected and synthesized based on hydrophilicity profiles. Two peptides represent sequences in the rat (and mouse) pro-NGF, one peptide (our peptide P3) represents a highly conserved region of the mature NGF protein (identical in humans, mice, rats, and chickens), two peptides are specific for the mature chicken NGF, and the remaining three peptides are specific for the mature rat NGF (each with only one amino acid substitution compared with corresponding segments of the mouse NGF). For immunization, the peptides were conjugated to keyhold limpet hemocyanin and used to produce antisera in rabbits. After bleeding, peptide-specific antibodies were purified on affinity columns prepared by coupling each of the synthetic peptides. The different peptide antisera and affinity-purified antibodies then were characterized by enzyme-linked immunoassay (ELISA) and immunohistochemistry of the male mouse submandibular gland, a rich exocrine source of NGF. ELISA analysis showed that all peptide antisera bound two to four orders of magnitude better than normal rabbit serum to a coat of their proper peptide. The higher binding was retained by the purified peptide antibodies compared with normal rabbit immunoglobulin. Specific tests, in which one peptide antiserum was checked against different peptide coats in the ELISA, also showed two to four orders of magnitude higher binding of antibodies to the proper synthetic peptide. The peptide antibodies also were tested for their ability to bind to native mouse beta NGF coated to the immunoplates. Only antibodies raised to the conserved P3 peptide recognized native NGF to an extent similar to that obtained with polyclonal anti-NGF antibodies. Conversely, P3 was well recognized by several different NGF antisera. Immunohistochemically, both peptide antisera against the pro-NGF stained the perinuclear cytoplasm in the basal part of the cells of the granulated convoluted tubules in the mouse submandibular gland.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1989

Development and regional expression of chicken neuroleukin (glucose-6-phosphate isomerase) messenger RNA.

Neuroleukin (NLK) is a protein identical with the glycolytic enzyme glucose-6-phosphate isomerase (GPI) that has been reported to support the survival of a subpopulation of neurons in embryonic dorsal root ganglia and spinal cord neurons in culture. In this report we have studied the developmental expression of NLK mRNA in the chick embryo in order to evaluate its possible role as a neurotrophic factor. The chicken gene encoding NLK was isolated by cross-hybridization to a mouse NLK cDNA clone. A DNA fragment from the chicken NLK gene with a 90% nucleotide sequence homology to mouse NLK cDNA encoding amino acids 310-355 was then used as a hybridization probe in a series of RNA-blots. In the entire embryo NLK mRNA was found already at embryonic day 3.5 (E3.5) and the level of expression was significantly decreased between E3.5 and hatching. Roughly similar levels of NLK mRNA were found in all tissues of the E8 embryo analyzed with the exception of the brain, which contained only low levels. When the developmental expression was analyzed in different tissues separately, NLK mRNA expression was found to decrease during development in the heart and bursa of Fabricius, whereas the level of mRNA in the brain showed a large increase shortly after hatching. The spinal cord and the pectoral and femoral muscles all showed high levels of NLK mRNA throughout development. In the adult chick, the highest levels of NLK mRNA were found in the muscle, brain, and kidney, where the NLK mRNA was estimated to account for approximately 0.1% of the total mRNA in these tissues. A widespread expression of NLK mRNA was observed in the adult brain with approximately similar levels in all brain regions tested. Similar results were also obtained when NLK mRNA expression was analyzed in adult rats. Our results show that developmental expression of the NLK gene is independently regulated in different tissues. The widespread and abundant expression of both the avian and rodent NLK gene is in accordance with its newly discovered identity as a glycolytic enzyme. Consequently, the developmental and adult pattern of NLK mRNA expression does not favour a specific trophic role for this protein in accordance with other known neurotrophic factors.

Animals↗
PubMed1989

Aged adrenal medullary tissue survives intraocular grafting, forms nerve fibers and responds to nerve growth factor.

Adrenal medullary tissue from aged (24 months old) and young adult (2 months old) rats was grafted to the anterior chamber of the eye of previously sympathectomized animals. Nerve growth factor (NGF) was administered by weekly bilateral intraocular injections. Five weeks postgrafting, irides were prepared as whole mounts and processed for Falck-Hillarp histochemistry for visualization of catecholamines. NGF appeared to partially prevent the reduction in volume that both old and young grafts underwent. In the presence of NGF, an extensive, dense fiber network, closely resembling the normal adrenergic innervation, was formed in the host irides by grafts from aged donors. The area of outgrowth from aged transplants without NGF treatment was as large as with NGF treatment but less dense. The reinnervation of irides by NGF-treated young adult grafts occupied a similar area as that seen with aged grafts, but the pattern of innervation was irregular, particularly close to the transplants. Transplants from young adult donors without NGF treatment generated a sparse, limited network of nerves in the irides. All grafts were tyrosine hydroxylase-, adrenaline-, and dopamine-beta-hydroxylase-immunoreactive in about the same proportion of cells, but the grafts from the young donors were smaller in size. We concluded that the ability of chromaffin cells to transform toward a neuronal phenotype, produce nerve fibers, and respond to exogenous NGF is maintained in aged adrenals.

Adrenal Medulla↗
PubMed1989

NGF treatment promotes development of basal forebrain tissue grafts in the anterior chamber of the eye.

The effects of nerve growth factor (NGF) on developing central cholinergic neurons were studied using intraocular grafts of rat fetal (E17) basal forebrain tissue. Prior to grafting, grafts were incubated in NGF or saline. Transplants were allowed to mature for six weeks, receiving weekly intraocular injections of NGF or saline. Measurements of NGF levels in oculo after one single injection showed that NGF slowly decreases in the anterior chamber fluid, and after one week, low but significant levels were still present in the eye. Following pretreatment with diisopropylfluorophosphate (DFP), the cholinergic neurons in the grafts were analyzed using three morphological markers: antibodies to cholineacetyltransferase (ChAT), antibodies to acetylcholinesterase (AChE Ab) and acetylcholinesterase histochemistry (AChE). The transplants grew well and became vascularized within the first week. The growth of the NGF-treated basal forebrain grafts was significantly enhanced as compared to the growth of the saline-treated grafts evaluated with repeated stereomicroscopical observations directly through the cornea of the ether-anaesthetized hosts. The NGF-treated grafts contained almost twice as many cholinergic neurons seen with all the cholinergic markers used, as the saline-treated grafts. However, there was no difference in cholinergic cell density between the two groups. The morphology and size of an individual cholinergic neuron was similar in the two groups. The fiber density as evaluated with AChE-immunohistochemistry did not change after NGF-treatment. The DFP-treatment did not seem to affect the AChE-immunoreactivity since an extensive fiber network was found, whereas almost no fibers were seen using conventional AChE histochemistry. We have demonstrated that in oculo transplantation of basal forebrain is a useful model for examining in vivo effects of NGF on central cholinergic function. The marked volume increase of NGF-treated grafts and the unchanged density of cholinergic cells and terminals suggests, that NGF increases the survival of not only developing cholinergic neurons, but possibly other non-cholinergic neurons and non-neuronal cells as well. These results support the notion that NGF acts as a neurotrophic factor on cholinergic and possibly non-cholinergic cells in the central nervous system.

Acetylcholinesterase↗
PubMed1989

Testicular atrophy and loss of nerve growth factor-immunoreactive germ cell line in rats exposed to n-hexane and a protective effect of simultaneous exposure to toluene or xylene.

Testicular and germ cell line morphology in rats were studied 2 weeks, 10 months and 14 months after cessation of a 61-day inhalation exposure to 1000 ppm n-hexane. Androgen biosynthetic capacity of testis, testosterone blood concentration, vas deferens morphology and noradrenaline (NA) concentration, epididymal sperm morphology, and fertility were also studied. Severe testicular atrophy involving the seminiferous tubules with loss of the nerve growth factor (NGF) immunoreactive germ cell line was found. Total loss of the germ cell line was found in a fraction of animals up to 14 months post-exposure, indicating permanent testicular damage. No impairment of androgen synthesis or androgen dependent accessory organs was observed. Simultaneous administration of 1000 ppm n-hexane and 1000 ppm toluene, or 1000 ppm n-hexane and 1000 ppm xylene, did not cause germ cell line alterations or testicular atrophy. Toluene and xylene were thus found to protect from n-hexane induced testicular atrophy.

Animals↗
PubMed1989

Detection of nerve growth factor and its mRNA by separate and combined immunohistochemistry and in situ hybridization in mouse salivary glands.

Intense labelling of secretory cells in the male mouse submandibular gland was observed after in situ hybridization using mouse nerve growth factor (NGF) cDNA probes. Under the same conditions, sparse less intensely labelled cells were also found in the sublingual gland. Hybridization to a chicken NGF cDNA probe gave weak labelling on the glands in accordance with a weak cross-hybridization between mouse NGF mRNA and chicken NGF cDNA probes, whereas no labelling was seen using pUC9 DNA as a hybridization probe. A combination of in situ hybridization and immunohistochemistry was also carried out on the same sections of submandibular gland. A good correlation was seen between actively synthesizing and intensely immunoreactive cells in the gland. The technique described here allows the detection of individual cells synthesizing relatively low levels of NGF. The combination of in situ hybridization and immunocytochemistry on the same section should be particularly useful in cases where NGF is transported away from its site of synthesis.

Animals↗
PubMed1989

Nerve growth factor can influence growth of cortex cerebri and hippocampus: evidence from intraocular grafts.

The effects of nerve growth factor and antiserum against nerve growth factor on cortical cholinergic projection areas in the central nervous system and cerebellum were evaluated using intraocular grafts of cortex cerebri, hippocampus and cerebellum in rat hosts receiving injections into the anterior chamber of the eye of nerve growth factor (at transplantation, 5 and 10 days after transplantation) or antiserum to nerve growth factor (every 5 days). The controls received cytochrome c or preimmune serum. Growth of grafts was followed by repeated observations directly through the cornea of the host using a stereomicroscope. Nerve growth factor-treated grafts of cortex cerebri and hippocampus grew significantly smaller as compared to the corresponding control grafts. In one experiment, growth of cytochrome c and saline-treated cortex cerebri was compared and no difference in growth was found. Growth of nerve growth factor-treated cerebellar grafts did not differ significantly from growth of cytochrome c-treated grafts. Morphological analysis using Nissl-staining, antibodies to glial acidic fibrillary protein to evaluate the degree of gliosis and antiserum to neurofilament as a neuronal marker did not reveal any marked differences between nerve growth factor- and cytochrome c-treated grafts. Cortical grafts receiving anti-nerve growth factor antiserum by injection or by immunizing host rats against nerve growth factor showed similar growth to the controls. Similarly, grafts of fetal hippocampus to rats immunized with nerve growth factor were not significantly different from grafts to host rats immunized with cytochrome c. We conclude that exogenous nerve growth factor affects the development of grafted cortex cerebri and hippocampus. The fact that these cortical areas stop growing earlier in the presence of nerve growth factor without the grafts showing evidence of disturbed glial or neuronal populations compared to control grafts indicates that nerve growth factor acts to induce overall/premature differentiation and maturation. The mechanism for this whether or not it is receptor-mediated and which cells are primarily affected by nerve growth factor is not yet known.

Animals↗
PubMed1989

NGF in CNS: experimental data and clinical implications.

The presence of beta-nerve growth factor (NGF) and its cell surface receptor (NGF-R) in the brain has been well established by a variety of experimental techniques in recent years. In particular, the molecular cloning of NGF and NGF-R as well as the development of sensitive two-site ELISA techniques for determining the levels of NGF and antibodies to NGF-R suitable for immunohistochemistry have led to rapid accumulation of data in this field from many laboratories. A main finding is the function of NGF in the cholinergic neurons of the basal forebrain, expressing NGF receptors and responding to the factor by increased activity of choline acetyltransferase, and the production of NGF in cortical areas and hippocampus comprising terminal areas for the cholinergic projections from the basal forebrain. In addition, findings suggest that additional neurons in the brain and spinal cord may utilize NGF, notably during development and possibly also after lesion of the adult CNS. Moreover, observations indicate that endogenous levels of NGF are lowered in the aged rat brain concomitant with losses of NGF-dependent neurons in the basal forebrain. The involvement of NGF in human neurodegenerative diseases is not established but the application of NGF to degenerating cholinergic neurons in Alzheimer patients may prove useful. A promising approach to achieve this goal is the production of biologically active, recombinant NGF.

Aging↗
PubMed1989

A cell line producing recombinant nerve growth factor evokes growth responses in intrinsic and grafted central cholinergic neurons.

The rat beta nerve growth factor (NGF) gene was inserted into a mammalian expression vector and cotransfected with a plasmid conferring resistance to neomycin into mouse 3T3 fibroblasts. From this transfection a stable cell line was selected that contains several hundred copies of the rat NGF gene and produces excess levels of recombinant NGF. Such genetically modified cells were implanted into the rat brain as a probe for in vivo effects of NGF on central nervous system neurons. In a model of the cortical cholinergic deficits in Alzheimer disease, we demonstrate a marked increase in the survival of, and fiber outgrowth from, grafts of fetal basal forebrain cholinergic neurons, as well as stimulation of fiber formation by intact adult intrinsic cholinergic circuits in the cerebral cortex. Adult cholinergic interneurons in intact striatum also sprout vigorously toward implanted fibroblasts. Our results suggest that this model has implications for future treatment of neurodegenerative diseases.

Acetylcholinesterase↗
PubMed1989

Screening for neurotrophic disturbances in amyotrophic lateral sclerosis.

Neurotrophic activities in human serum and post-mortem muscle and spinal cord of possible relevance to pathophysiological mechanisms in amyotrophic lateral sclerosis (ALS) were studied. Tests included in vitro assays for nerve fibre outgrowth from sympathetic ganglia and for survival promotion of dissociated ciliary neurons, both types of neurons, of chicken embryo origin. Extracts of postmortem biceps muscle promoted survival of ciliary neurons in a dose-dependent manner. Half-maximum effect was found at a protein concentration of about 450 micrograms/ml for both ALS and control muscle. Ventral horn extracts were about 5 times as efficient as muscle in promoting neuron survival, again with no differences seen between control and ALS samples. Sera from patients suffering from ALS as well as normal sera did not enhance survival of ciliary neurons to any considerable extent, nor did they induce fibre outgrowth from sympathetic ganglia. Both groups of sera, if present above 5% in the medium, suppressed fibre outgrowth induced by added nerve growth factor (NGF). Sera from some of the ALS patients impaired survival in dissociated ciliary neurons supported by a trophic activity in choroid extract. The results do not indicate major neurotrophic deficits as the cause of ALS disease but suggest that a neurotoxic mechanism may be involved.

Aged↗
PubMed1988

Expression of the beta-nerve growth factor gene in hippocampal neurons.

In situ hybridization with complementary DNA probes for nerve growth factor (NGF) was used to identify cells containing NGF messenger RNA in rat and mouse brain. The most intense labeling occurred in hippocampus, where hybridizing neurons were found in the dentate gyrus and the pyramidal cell layer. The neuronal identity of NGF mRNA-containing cells was further assessed by a loss of NGF-hybridizing mRNA in hippocampal areas where neurons had been destroyed by kainic acid or colchicine. RNA blot analysis also revealed a considerable decrease in the level of NGF mRNA in rat dentate gyrus after a lesion was produced by colchicine. This lesion also caused a decrease in the level of Thy-1 mRNA and an increase in the level of glial fibrillary acidic protein mRNA. Neuronal death was thus associated with the disappearance of NGF mRNA. These results suggest a synthesis of NGF by neurons in the brain and imply that, in hippocampus, NGF influences NGF-sensitive neurons through neuron-to-neuron interactions.

Animals↗
PubMed1988

Differential outgrowth of retinal neurites on purified extracellular matrix molecules.

Organotypic cultures of the embryonic retina were used to study the influence of extracellular matrix molecules on neurite elongation during development of the central nervous system. Microexplants from the chick retina (embryonic day 6) were grown in medium containing appropriate trophic support on purified matrix molecules adsorbed to plastic at various concentrations. The maximum neurite length obtained on each type of substratum was measured on day 4 of culture. No fiber outgrowth occurred on substrata of vitronectin or a hyaluronate-binding chondroitin sulfate proteoglycan. In contrast, neurite elongation was strongly promoted on laminin in a dose-dependent manner. Fibronectin elicited a neurite outgrowth corresponding to about one-third the length of the outgrowth on laminin. A 31,000-dalton fibronectin fragment representing the heparin-binding domain elicited neurite elongation comparable to that promoted by the intact fibronectin molecule. Other isolated domains of fibronectin, including the 105,000-dalton "cell-binding" domain, did not allow neurite outgrowth. Furthermore, preincubation of fibronectin substratum with antibodies to the heparin-binding fibronectin fragment entirely prevented outgrowth. Fiber outgrowth was also evoked on substrata of platelet factor 4, a protein binding heparan sulfate. Adding increasing concentrations of heparin progressively inhibited the neurite extension on laminin, whereas similar addition of soluble chondroitin sulfate proteoglycan had no effect. The results indicate that growing retinal neurites show strong preference for laminin versus fibronectin. Moreover, the outgrowth-promoting activity of both cell adhesion proteins seems to be localized to their heparin-binding regions. It is suggested that during development of the visual system, elongating retinal neurites can actively discriminate between different extracellular molecules by a mechanism that may involve participation of cell surface heparan sulfate proteoglycans.

Animals↗
PubMed1988

Fate of intraocular chromaffin cell suspensions: role of initial nerve growth factor support.

Adrenal medullary tissue from adult rats was dissociated into cell suspensions and injected into the anterior chamber of the eye, where the cells were made to attach to the previously sympathectomized irides with the use of fibronectin. Short- and long-term survival of the chromaffin cells was examined in whole mounts of irides using Falck-Hillarp fluorescence histochemistry or indirect immunohistochemistry with antibodies against adrenaline and dopamine-beta-hydroxylase (DBH). After 6 days in oculo all cells were immunoreactive for adrenaline; almost none displayed processes even if beta-nerve growth factor (NGF) was given at grafting. One month after weekly intraocular injections of NGF, many cells were surrounded by nerve fiber networks and all cells were DBH-immunoreactive. Eight months postgrafting and 7 months after the last injection of NGF almost the entire iris was reinnervated and resembled a normal, sympathetically innervated iris. Both at 1 and 8 months, chromaffin cells, ganglion cells and transitional cell forms (chromaffin cells transforming towards ganglion-like cells) were found in irides from the NGF-treated eyes. The number of ganglion cells was remarkably increased with time by NGF, while the number of chromaffin cells decreased compared to controls. A single treatment with NGF at grafting had no marked effects as examined up to 3 months; at this time there was a certain outgrowth of nerve terminals, which, however, was not as pronounced as 1 month after repeated NGF injections. In conclusion, it is shown that some cells in a chromaffin cell suspension attach to the iris, transform to ganglion cells after an induction with exogenous NGF, and reinnervate the sympathetically denervated iris. Such cells remain ganglion-like in character and continue to form processes even after cessation of exogenous NGF treatment.

Adrenal Medulla↗