Subunit Of Nerve Growth Factor Research Articles

Effect of the Camelid's Seminal Plasma Ovulation-Inducing Factor/β-NGF: A Kisspeptin Target Hypothesis.

Female mammals are classified into spontaneous and induced ovulators based on the mechanism eliciting ovulation. Ovulation in spontaneous species (e.g., human, sheep, cattle, horse, pigs, and most rodents) occurs at regular intervals and depends upon the circulating estradiol. However, in induced ovulators (e.g., rabbits, ferrets, cats, and camelids), ovulation is associated with coitus. In the later, various factors have been proposed to trigger ovulation, including auditory, visual, olfactory, and mechanic stimuli. However, other studies have identified a biochemical component in the semen of induced ovulators responsible for the induction of ovulation and named accordingly ovulation-inducing factor (OIF). In camelids, intramuscular or intrauterine administration of seminal plasma (SP) was shown to induce the preovulatory luteinizing hormone (LH) surge followed by ovulation and subsequent formation of corpus luteum. Recently, this OIF has been identified from SP as a neurotrophin, the β subunit of nerve growth factor (β-NGF). β-NGF is well known as promoting neuron survival and growth, but in this case, it appears to induce ovulation through an endocrine mode of action. Indeed, β-NGF may be absorbed through the endometrium to be conveyed, via the blood stream, to the central structures regulating the LH preovulatory surge. In this review, we provide a summary of the most relevant results obtained in the field, and we propose a working hypothesis for the central action of β-NGF based on our recent demonstration of the presence of neurons expressing kisspeptin, a potent stimulator of GnRH/LH, in the camel hypothalamus.

IFRD1 Is a Candidate Gene for SMNA on Chromosome 7q22-q23

We have established strong linkage evidence that supports mapping autosomal-dominant sensory/motor neuropathy with ataxia (SMNA) to chromosome 7q22-q32. SMNA is a rare neurological disorder whose phenotype encompasses both the central and the peripheral nervous system. In order to identify a gene responsible for SMNA, we have undertaken a comprehensive genomic evaluation of the region of linkage, including evaluation for repeat expansion and small deletions or duplications, capillary sequencing of candidate genes, and massively parallel sequencing of all coding exons. We excluded repeat expansion and small deletions or duplications as causative, and through microarray-based hybrid capture and massively parallel short-read sequencing, we identified a nonsynonymous variant in the human interferon-related developmental regulator gene 1 (IFRD1) as a disease-causing candidate. Sequence conservation, animal models, and protein structure evaluation support the involvement of IFRD1 in SMNA. Mutation analysis of IFRD1 in additional patients with similar phenotypes is needed for demonstration of causality and further evaluation of its importance in neurological diseases.

Dermal fibroblasts from different layers of human skin are heterogeneous in expression of collagenase and types I and III procollagen mRNA.

We have previously shown that the expression of collagenase mRNA and activity are suppressed in fibroblasts derived from postburn hypertrophic scar. Although it is known that differential synthesis of collagen and collagenase in postburn hypertrophic scar is one of the main reasons for the excessive accumulation of collagen, it is not clear why the expression of collagenase in hypertrophic scar fibroblasts is suppressed relative to that in normal fibroblasts. In this study, we hypothesized that dermal fibroblasts from deeper layers of skin, which normally migrate toward the wound site, have a different capacity to express key extracellular matrix proteins such as collagenase and types I and III procollagen. To test this hypothesis, we established four different pairs of hypertrophic and site-matched normal cell strains from four different patients with postburn hypertrophic scarring. In another set of experiments, ten different cell strains from two normal human skin samples that were horizontally sectioned into five different layers (layers 1-5 correspond to upper to deeper layers, respectively) were established in culture. Cells at the same passages were harvested, total RNA was extracted, and Northern analysis was conducted to determine the level of collagenase and types I and III procollagen mRNA expression in each cell strain. The results of Northern blot analysis showed two transcripts each for the pro alpha1(I) collagen chain (5.8 and 4.8 kb) and for the pro alpha1(III) collagen chain (5.4 and 4.8 kb) in all cell strains examined. The intensity of the pro alpha1(I) chain of type I procollagen mRNA varied, ranging from 50.8 to 137.1 and 44.4 to 131.5 densitometry units among either normal or hypertrophic scar cells examined, respectively. Similarly, the relative quantity of type III procollagen transcript also varied, ranging from 10.4 to 91.1 and 28.7 to 116.1 among normal and hypertrophic scar cells, respectively. When five different skin layers from each tissue sample were evaluated for functional cell heterogeneity, the results showed a marked variation in expression of mRNA for type I and III procollagen. Although the level of collagenase mRNA also varied among different cell strains examined, the expression of collagenase mRNA was lower in fibroblasts derived from deeper layers of each skin sample. In conclusion, cells from different layers of normal skin samples are heterogeneous in their constitutive expression of some key extracellular matrix components such as collagenase and types I and III procollagen.

Nerve growth factor accelerates wound healing in diabetic mice

Patients with diabetic neuropathy have reduced numbers of cutaneous nerves, which may contribute to an increased incidence of nonhealing wounds. Nerve growth factor (NGF) has been reported to augment wound closure. We hypothesized that topical 2.5S NGF, a biologically active subunit of the NGF polymer, would accelerate wound repair, augment nerve regeneration, and increase inflammation in excisional wounds in diabetic mice. A full‐thickness 6‐mm punch biopsy wound was created on the dorsum of C57BL/6 J‐m + Leprdb mice (db/db) and heterozygous (db/–) littermates and treated daily with normal saline or 2.5S NGF (1 µg/day or 10 µg/day) on post‐injury days 0–6. Time to closure, wound epithelialization, and degree of inflammation were compared using a Student's t‐test. Color subtractive‐computer‐assisted image analysis was used to quantify immunolocalized nerves in wounds. Non‐overlapping (20×) digital images of the wound were analyzed for nerve profile counts, area density (number of protein gene product 9.5 positive profiles per unit dermal area) and area fraction (protein gene product 9.5 positive area per unit dermal area). Healing times in db/db mice decreased from 30 days in normal saline‐treated mice to 26 days in mice treated with 1 µg/day NGF (p < 0.05) and 24 days in mice treated with 10 µg/day NGF (p < 0.02). A similar trend in db/– mice was not significant. NGF treatment augmented epithelialization in the db/db mice (p < 0.05). Histological evaluation of inflammation in healed wounds showed no statistical difference between treatment groups. Total nerve number, area density, and area fraction were increased in NGF‐treated wounds at 14, 21, and 35 days (p < 0.05). The 2.5 NGF subunit may improve wound closure kinetics by promoting epithelialization and nerve regeneration. Further studies to determine the role of nerves in wound repair are warranted.

Regulation by Androgen of Levels of the β Subunit of Nerve Growth Factor and Its mRNA in Selected Regions of the Mouse Brain

Our previous studies showed that the concentration of the beta subunit of nerve growth factor (beta-NGF) in nervous tissues is higher in male than in female mice. To identify the brain regions that are affected by androgens, the amounts of beta-NGF protein and its mRNAs were measured in male, female, and castrated male CD-1 mice and testicular feminization mice at 3-4 months of age. Among tissues examined, the hypophysis of males contained the highest average concentration of beta-NGF protein. In most regions of the brain, individual levels were more variable in males than in females. However, after the castration, such variations in beta-NGF levels disappeared. Average levels of beta-NGF protein in males were higher in the cerebellum (eightfold higher), olfactory bulb (12-fold higher), hypothalamus (sixfold higher), and hypophysis (72-fold higher) than those in corresponding regions of females. No significant differences were observed in levels of beta-NGF protein in the hippocampus, cerebral cortex, striatum, septum, and brainstem. The castration of male mice caused a reduction in levels of beta-NGF protein in the hypothalamus and hypophysis, but not in the cerebellum and olfactory bulb, to the female levels. The concentrations of beta-NGF protein in testicular feminization mice were similar to those in female CD-1 mice in all regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve growth factor (NGF) in cerebrospinal fluid (CSF) from patients with various neurological disorders.

It has been recently shown that NGF is not only involved in the survival and development of sympathetic and neural crest-derived sensory neurons, but also in some mechanisms of the immune system. For this reason, we studied the content of NGF in CSF samples from patients with diseases in which neuroimmunological mechanisms seem to be involved (multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer disease, chronic relapsing polyradiculoneuritis, Guillain-Barré syndrome, and tumors of the nervous system), as well as from a number of normal control subjects. We setup an ELISA aimed at the beta subunit of NGF, obtaining good validation tests and a detection limit of 28 pg beta NGF per ml. None of the samples was found to contain detectable levels of NGF and, when a concentration method for sample enrichment was used, only one patient was NGF-positive. This suggests that NGF is probably not involved in the neuroimmunological mechanisms underlying some inflammatory and degenerative diseases of the nervous system.

Expression of NGF in sympathetic neurons leads to excessive axon outgrowth from ganglia but decreased terminal innervation within tissues

The effects of nerve growth factor (NGF) on sympathetic axon growth were investigated by generating transgenic mice in which the β subunit of NGF was expressed in sympathetic neurons using the human dopamine β-hydroxylase (DBH) promoter. In DBH-NGF mice, the sympathetic trunk and nerves growing to peripheral tissues were enlarged and contained an increased number of sympathetic fibers. Although sympathetic axons reached peripheral tissues, terminal sympathetic innervation within tissues was decreased in DBH-NGF mice. This effect could be reversed in the pancreas by overexpression of NGF in pancreatic islets. The observations are consistent with a model in which NGF gradients are not required to guide sympathetic axons to their targets, but are required for the establishment of the normal density and pattern of sympathetic innervation within target tissues.

Arginine specific endopeptidases modify the aggregation properties of a synthetic peptide derived from Alzheimer beta/A4 amyloid.

A synthetic peptide corresponding to the first 28 amino acids of the Alzheimer disease amyloid beta/A4 peptide (3.2 kDa) aggregated to a high molecular weight (15 kDa) on SDS/urea polyacrylamide gels. Proteinase K, V8 protease, trypsin, and endopeptidase Lys-C readily degraded the aggregate. By contrast, when digested by endopeptidase Arg-C, a new polypeptide aggregate of higher molecular weight (16 kDa) was observed on denaturing gels without degraded smaller products. The new aggregate was comprised of three peptides: an intact beta/A4(1-28) and partially degraded peptides beta/A4(1-5) plus beta/A4(6-28). The results were confirmed by treatment of beta/A4 with other arginine-specific proteases: the gamma subunit of nerve growth factor and clostripain. The results indicate that arginine-specific proteases, including a growth factor processing enzyme, can nick aggregated beta/A4(1-28) amyloid and alter the configuration to produce a more complex aggregated form. If similar highly specific proteolytic mechanisms occur in the Alzheimer disease brain, the processing may promote the formation of high molecular weight aggregates that contribute to the development of relatively insoluble senile plaque core protein.

Limbic Seizures Increase Neuronal Production of Messenger RNA for Nerve Growth Factor

Nerve growth factor (NGF) produced by telencephalic neurons provides critical trophic support for cholinergic neurons of the basal forebrain. In situ hybridization and nuclease protection analyses demonstrate that limbic seizures dramatically increase the amount of messenger RNA for NGF in the neurons of the hippocampal dentate gyrus within 1 hour of seizure onset and in broadly distributed neocortical and olfactory forebrain neurons some hours later. The increased messenger RNA species is indistinguishable from messenger RNA for transcript B of the beta subunit of NGF from mouse submandibular gland. Thus, the expression of a known growth factor is affected by unusual physiological activity, suggesting one route through which trophic interactions between neurons in adult brain can be modified.

Marcophate colony‐stimulating factor in nerve growth factor preparations

Following a report that nerve growth factor preparations have granulocyte-colony-stimulating activity, we investigated the presence of colony-stimulating factors in 7s mouse submaxillary nerve growth factor and its subunits. Macrophage colonies were formed in mouse bone marrow cultures after exposure to preparations of 7s nerve growth factor, the gamma subunit, and, to a small extent, the alpha subunit; the beta subunit, which is responsible for the nerve growth function, did not stimulate colony growth. Furthermore, the esteropeptidase activity of the gamma subunit was not detected in preparations of macrophage colony-stimulating factor purified from the giant cell tumor (GCT) cell line. Immunoprecipitation of radiolabeled gamma subunit with a polyclonal antibody to L-cell macrophage colony-stimulating factor showed a protein band that could represent the gamma subunit of nerve growth factor. Separation of the macrophage activity from the esteropeptidase activity of the gamma subunit was accomplished on the basis of molecular size. Thus, macrophage colony-stimulating factor was a contaminant of nerve growth factor produced by the mouse submaxillary gland and copurified with the gamma subunit.

Induction of a neural phenotype in a serotonergic endocrine cell derived from the neural crest

The thyroid parafollicular cell is an endocrine cell derived from the neural crest that stores 5-hydroxytryptamine (5-HT). In common with serotonergic neurons, but in contrast to 5-HT-storing cells that are not neurectodermal derivatives, parafollicular cells also contain a specific 5-HT binding protein. Despite this similarity to serotonergic neurons, parafollicular cells in situ were found to express an endocrine phenotype with few neural characteristics. Thus, the cells costore 5-HT with calcitonin, not calcitonin gene-related peptide (CGRP), which is the product of the calcitonin gene expressed in neurons, and they do not contain neurofilaments. The ability of adult parafollicular cells to respond to microenvironmental perturbations by expressing neuronal characteristics was examined. Sheep thyroid glands were dissociated, and parafollicular cells were purified by affinity chromatography. The purified parafollicular cells were grown in culture on a variety of substrates in the presence or absence of the beta subunit of nerve growth factor (beta-NGF). Parafollicular cells survived in culture for at least a week but retained a roughly spherical shape. Nevertheless, a subset of the cultured parafollicular cells began to display CGRP immunoreactivity. The addition of beta-NGF to the cultured parafollicular cells induced a number of them to extend neurites and increased the proportion of cells in which CGRP immunoreactivity could be found. Neurite-bearing parafollicular cells appeared not to survive for more than 2 d. While their survival was not enhanced when they were grown on collagen, polylysine, laminin, or reconstituted basal lamina, parafollicular cells that had extended neurites in response to beta-NGF survived for at least a week when cocultured with an explant of aneuronal chick hindgut. The effect of the gut was local and only those neurite-bearing parafollicular cells that were growing in direct contact with the explant survived. The thyroid parafollicular cell therefore resembles another crest-derived endocrine cell, the adrenal chromaffin cell, in being able to manifest neural properties in culture. For the parafollicular cell these neural properties include the processing of RNA encoded by the calcitonin gene to express CGRP and neurite outgrowth in response to beta-NGF.

Synthesis and secretion of a nerve growth-stimulating factor by neonatal mouse astrocyte cells in vitro.

Neonatal mouse astroglial cells cultured in a serum-free medium synthesize and secrete a trophic growth factor which resembles nerve growth factor (NGF). The NGF-like factor reacts with antiserum to beta subunit of NGF (beta-NGF) and, after labeling with [35S] cystine, migrates similarly to purified mouse beta-NGF in SDS polyacrylamide gel electrophoresis and Sephadex G-100 gel filtration systems. The astrocyte cell NGF-like factor displays beta-NGF-like neurite growth-promoting activity for the clonal rat pheochromocytoma (PC-12) cell line and this bioactivity is blocked by beta-NGF antiserum. These results indicate that NGF-like factor synthesized and secreted by astroglial cells, is similar, if not identical, to beta-NGF from the mouse submandibular gland and further support a potential role for NGF in the central nervous system.

Assignment of the gene for the beta subunit of thyroid-stimulating hormone to the short arm of human chromosome 1.

The chromosomal locations of the genes for the beta subunit of human thyroid-stimulating hormone (TSH) and the glycoprotein hormone alpha subunit have been determined by restriction enzyme analysis of DNA extracted from rodent-human somatic cell hybrids. Human chorionic gonadotropin (CG) alpha-subunit cDNA and a cloned 0.9-kilobase (kb) fragment of the human TSH beta-subunit gene were used as hybridization probes in the analysis of Southern blots of DNA extracted from rodent-human hybrid clones. Analysis of the segregation of 5- and 10-kb EcoRI fragments hybridizing to CG alpha-subunit cDNA confirmed the previous assignment of this gene to chromosome 6. Analysis of the patterns of segregation of a 2.3-kb EcoRI fragment containing human TSH beta-subunit sequences permitted the assignment of the TSH beta-subunit gene to human chromosome 1. The subregional assignment of TSH beta subunit to chromosome 1p22 was made possible by the additional analysis of a set of hybrids containing partially overlapping segments of this chromosome. Human TSH beta subunit is not syntenic with genes encoding the beta subunits of CG, luteinizing hormone, or follicle-stimulating hormone and is assigned to a conserved linkage group that also contains the structural genes for the beta subunit of nerve growth factor (NGFB) and the proto-oncogene N-ras (NRAS).

Protease nexin. Properties and a modified purification procedure.

The present paper describes chemical and functional properties of protease nexin, a serine protease inhibitor released from cultured human fibroblasts. It is shown that protease nexin is actually synthesized by fibroblasts and represents about 1% of their secreted protein. Analysis of the amino acid composition of purified protease nexin indicates that it is evolutionarily related to antithrombin III and heparin cofactor II. Protease nexin contains approximately 6% carbohydrate, with 2.3% amino sugar, 1.1% neutral sugar, and 3.0% sialic acid. The Mr calculated from equilibrium sedimentation analysis is 43,000. Protease nexin is a broad specificity inhibitor of trypsin-like serine proteases. It reacts rapidly with trypsin (kassoc = 4.2 +/- 0.4 X 10(6) M-1 s-1), thrombin (kassoc = 6.0 +/- 1.3 X 10(5) M-1 s-1), urokinase (kassoc = 1.5 +/- 0.1 X 10(5) M-1 s-1), and plasmin (kassoc = 1.3 +/- 0.1 X 10(5) M-1 s-1), and slowly inhibits Factor Xa and the gamma subunit of nerve growth factor but does not inhibit chymotrypsin-like proteases or leukocyte elastase. In the presence of heparin, protease nexin inhibits thrombin at a nearly diffusion-controlled rate. Two heparin affinity classes of protease nexin can be detected. The present characterization pertains to the fraction of protease nexin having the higher affinity for heparin. The low affinity material, which is the minor fraction, is lost during purification.

Chromosomal locations of the human and mouse genes for precursors of epidermal growth factor and the beta subunit of nerve growth factor.

DNA probes for pre-pro-epidermal growth factor (EGF) and the precursor of the beta subunit of nerve growth factor (NGF) were used to chromosomally map human and mouse EGF and NGF genes in panels of human-mouse and mouse-Chinese hamster somatic cell hybrids. The EGF and NGF genes were mapped to human chromosomes 4 and 1, respectively, by using human-mouse cell hybrids. A combination of regional mapping using a chromosome 1 translocation and comparative gene mapping suggests that the human NGF gene is in the p21-p22.1 region of chromosome 1. In mouse-Chinese hamster cell hybrids, both genes were assigned to mouse chromosome 3. A knowledge of the chromosomal assignment of these genes should help in our understanding of their regulation and role in development and disease.

Linkage relationships of the gene for the beta subunit of nerve growth factor (NGFB) with other chromosome 1 marker loci.

Linkage relationships of the gene for the beta subunit of nerve growth factor (NGFB) with other chromosome 1 marker loci.

The human gene for the beta subunit of nerve growth factor is located on the proximal short arm of chromosome 1.

Fragments of the recently cloned human gene for the beta subunit of nerve growth factor (beta-NGF) were used as hybridization probes in analyzing two sets of rodent-human somatic cell hybrids for the presence of human beta-NGF sequences. Results from the first set of hybrids assigned the human beta-NGF gene to chromosome 1 and ruled out the presence of sequences of comparable homology on any other chromosome. With the second set of hybrids, which contained seven different, but overlapping, regions of chromosome 1, the NGF locus was mapped to band 1p22.

Increased serum levels of nerve growth factor in von Recklinghausen's disease.

Serum samples from patients with peripheral von Recklinghausen's disease were studied by competition radioreceptor assays and radioimmunoassays for biologically active 2.5S subunit of nerve growth factor (NGF). These specimens were fractionated at low pH to partially purify a 2.5S NGF-like activity that specifically competes for the binding of radioisotope-labeled 2.5S NGF to its receptor on human melanoma cells. In seven patients with peripheral neurofibromatosis, statistically significant elevations of this 2.5S NGF competing activity were found: approximately 85% of the patients tested had competing activities outside the 95% confidence limits of the normal population. No elevations of competing activity were observed when these serum fractions were examined by a specific radioimmunoassay for purified 2.5S NGF. These conflicting observations may suggest the existence of two molecular species demonstrating NGF-like activity: one sharing antigenic determinants with mouse 2.5S NGF and the other antigenically unrelated. We identified an NGF binding activity of high molecular weight in human serum. This activity is not elevated in the serum of patients with peripheral neurofibromatosis.

Evidence that the arginine esteropeptidase (gamma) subunit of nerve growth factor can activate inactive renin

The beta and gamma subunits of nerve growth factor were isolated and tested for inactive-reninactivating activity using human amniotic fluid as a convenient source of inactive renin. Only the gamma subunit, which is an arginine esteropeptidase, could activate inactive renin. The role of this enzyme in sites containing both it and inactive renin warrants investigation.

Effect of thyroxine on nerve growth factor concentration in neonatal mouse brain

The effect of T4 administration on central nervous system nerve growth factor (NGF) concentration was studied in 12 day old mice. Neonatal mice received T4 (0.4 or 1.6 ug/g body weight) daily for 11 days after birth. On the 12th day, NGF concentration was measured in cerebral cortex, cerebellum and brain stem using a specific radioimmunoassay for the biologically active beta subunit of NGF; this assay is sensitive to 10–15 pg NGF. T4 administration caused a dose related decrease in mean body weight to 86% and 77% of control body weight for the low and high T4 doses respectively. Mean cerebral cortex and cerebellum weights (corrected for body weight) were significantly increased in the T4 treated mice. Brain stem weight, however, was significantly decreased. T4 (0.4 ug/g body weight) caused a significant increase in mean NGF concentration (pg/mg protein) in cerebral cortex (26%; p <0.02) cerebellum (54%; p <0.001) and brain stem 42%; p <0.01) compared to control mice. The higher dose of T4 (1.6 ug/g body weight) also caused significant increases in mean NGF concentrations in the three brain areas but the increases were not statistically greater than the increments observed after the 0.4 ug/g T4 dose. Brain maturation in neonatal rodents occurs largely during the first 2–4 weeks of postnatal life; and thyroid hormones are essential for the orderly and sequential progress of this maturation. Thyroid hormone excess accelerates histological and biochemical maturation of the immature central nervous system (1–3) (CNS) whereas thyroid hormone deficiency results in significant delays in the attainment of biochemical and histological landmarks of maturation (1, 2, 4). The mechanism of this thyroid hormone effect is unknown. Specific nuclear binding sites for thyroid hormones are present in high concentrations in the immature CNS (5, 6). However thyroid hormone effects observed in other thyroid hormone responsive tissues, i.e., liver and kidney, following thyroid hormone binding to specific nuclear receptors are not observed in the immature brain (7). We have previously shown that thyroxine (T4) administration to mature male mice significantly increases nerve growth factor (NGF) content and concentration in cerebral cortex, cerebellum and brain stem (8). The present studies were conducted to determine whether thyroxine influences CNS NGF during the period of neonatal brain maturation to support the hypothesis that the thyroid hormone effects on CNS maturation may be mediated by NGF.