Isolation of human nerve growth factor from placental tissue

Nerve growth factor (NGF) has been isolated from human placental tissue. Using the chicken embryo dorsal root ganglia assay, we determined levels of NGF activity for the amnion, placental cotyledons, cord serum, fetal serum, and maternal serum. The highest levels of NGF activity were measured in placental cotyledons. After homogenization and centrifugation of the placental cotyledons, the supernatant was sequentially chromatographed, at neutral pH, on Sephadex G-100, DEAE-11, and Sephadex G-150. A high-molecular-weight protein fraction (150,000), which contained all the biological activity, was isolated in this fashion. Analytical isoelectric focusing of this fraction revealed a basic protein component (pI 9.5) of the high-molecular-weight species. Assays for NGF activity of all protein components separated by analytical isoelectric focusing showed that NGF activity was associated only with the basic protein component. Correspondingly, preparative isoelectric focusing of the high-molecular-weight species yielded a basic protein with very high biological activity (1–3 ng per biological unit) that was immunochemically active against rabbit IgG made against mouse -NGF.To whom reprint requests should be addressed.     

Nerve growth factor has both mitogenic and antimitogenic activity

The present study demonstrates that nerve growth factor (NGF) possesses both antimitogenic and mitogenic activities. To this end, we have employed clonal PC12 rat pheochromocytoma cells and two PC12 variant sublines, U2 and U7. When PC12 cells are exposed to NGF in culture media that are otherwise either permissive (15% serum) or restrictive (1% serum) for proliferation, neuronal differentiation occurs and mitosis ceases. Variant lines of PC12 cells have been selected that continue to proliferate in the presence of NGF in permissive medium but which nevertheless retain NGF receptors and certain NGF responses. In contrast to the parent PC12 cells, when such variants were exposed to NGF in growth-restrictive media, cell proliferation was markedly stimulated. The mitogenic activity of NGF was detectable at 0.1 ng/ml (4 pM) and was maximal at 3 ng/ml (100 pM). Possible contamination of the NGF preparation by epidermal growth factor (EGF) or mitogenic proteolytic enzymes was ruled out by the use of anti-EGF and diisopropylfluoro-phosphate, respectively. These findings show that NGF shares the capacity to stimulate cell division with a variety of other peptide hormones and suggest that the mitogenic activity of NGF could play a role in development of the peripheral nervous system as well as in promotion of in vivo growth of certain neural crest-derived neoplasms.     

Nerve growth factor in serum of children with systemic lupus erythematosus is correlated with disease activity

Nerve growth factor (NGF) is a neurotrophic factor, which is expressed both in the nervous system and in peripheral organs. NGF is also present in mast cells, and in B- and T-lymphocytes, and may play a role in the immune cell development and differentiation. Various cytokines have been shown to affect NGF expression, and NGF is elevated in inflammation and in some autoimmune diseases. Here we have studied NGF concentrations in serum of pediatric patients with systemic lupus erythematosus (SLE) using a two-site enzyme-linked immunosorbent assay (ELISA). We have further correlated the levels of NGF to the inflammatory state of the disease. The mean value of serum NGF in SLE patients was significantly increased compared with controls (3346 vs 627pg/ml). There was a correlation between the activity of SLE and the levels of NGF. The results show that NGF is elevated in childhood SLE and that the levels are correlated with disease activity. The present results suggest that NGF may play a role in the pathogenesis of SLE and may have a prognostic value in evaluating the course of the disease and in outlining the medication.     

Cross-Linked 7S Nerve Growth Factor Is Biologically Inactive

Abstract: The 7S nerve growth factor complex was cross-linked with dimethylsuberimidate in a pH jump reaction and the cross-linked product of highest molecular weight was separated from non-cross-linked subunits by gel filtration at acid pH. The amount of the cross-linked 7S nerve growth factor generated was reduced by exposure of the complex to pH 9.5, where it dissociates. The cross-linked 7S nerve growth factor migrated as a single species on electrophoresis under denaturing and reducing conditions and had a molecular weight, 132,000, identical to that of 7S nerve growth factor. All three subunits of the complex were released on deamidination. The biological activity of the cross-linked 7S nerve growth factor complex was less than 0.3% that of native βNGF, suggesting that the biological activity of the latter is completely inhibited by interactions with other subunits in the complex.     

Characterization of a factor that promotes neurite outgrowth: evidence linking activity to a heparan sulfate proteoglycan

Rat sympathetic neurons, plated onto extracellular matrix produced by cultured bovine corneal endothelial cells, rapidly extended neurites in the absence of nerve growth factor (NGF). The response was unaffected by antiserum to NGF. Rapid outgrowth also occurred when sympathetic neurons were plated onto polylysine-coated surfaces that had been exposed to serum-free medium conditioned by corneal endothelial cells (CMSF). A response was seen even when the neurons were cultured without serum. When plated onto a polylysine-coated dish treated with CMSF over half its surface, only the neurons on the treated half extended neurites. The active factor in CMSF was destroyed by trypsin, acid (pH 1.6), base (pH 12.7), or heating to 80 degrees C; it was stable to heating to 60 degrees C, collagenase, deoxyribonuclease, and neuraminidase. The factor elutes just after the void volume of a Sepharose 6B column. In associative cesium chloride gradients, it sediments as a peak centered at a density of 1.36-1.37, corresponding to a peak of material that can be biosynthetically labeled with [35S]sulfate or [3H]leucine. Material from this fraction was inactivated by heparinase, but not chondroitinase ABC, implying that a heparin sulfate proteoglycan is essential for the factor's activity. Inactivation by contaminants in the heparinase preparation was ruled out. Further purification indicated that the active factor may exist as an aggregate containing a heparin sulfate proteoglycan and other molecules. CMSF also promoted neurite outgrowth by other types of neurons. Furthermore, a variety of cell types were shown to produce factors similar to that in CMSF.     

Nerve growth factor (NGF) induces sprouting of specific neurons of the snail, Lymnaea stagnalis

Nerve growth factor (NGF) was examined for its ability to elicit sprouting by adult molluscan neurons. Motoneurons and interneurons (but not neurosecretory cells) from Lymnaea exhibited a sprouting response to murine 2.5S NGF in defined medium with a half-maximal response at about 150 ng/mL. Furthermore, an NGF antiserum blocked sprouting by all normally responsive neurons. We tested whether an NGF-like molecule is a component of conditioned medium (CM) by attempting to preabsorb its sprout-inducing activity with NGF antiserum. Treatment of CM with immune (but not nonimmune) serum largely blocked the response of motoneurons, but not that of neurosecretory cells, to CM. We conclude that NGF exerts neurotrophic activity on specific adult Lymnaea neurons, and suggest the possibility that an NGF-like molecule may exist in the molluscan nervous system.     

Lack of correlation between 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and lovastatin resistance in nerve growth factor treated PC-12 cells

Summary 1. The relationships among the mevalonic acid (MVA) forming enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (CoA) reductase, cell growth and differentiation, and the cytotoxic effects of the reductase inhibitor lovastatin were studied in PC-12 cells, exposed to growth factors.2. When added individually, nerve growth factor (NGF), basic fibroblast growth factor, and epidermal growth factor induce an increase in HMG-CoA reductase activity in cells grown in serum-containing medium. In the presence of serum, the effect of NGF on HMG-CoA reductase is persistent.3. Short-term serum starvation and long-term NGF treatment, in combination, have an additive effect, resulting in a high reductase activity.4. Unlike serum and MVA, which downregulate levels of HMG-CoA reductase by accelerating its degradation, NGF upregulates reductase by slowing the rate of its degradation. This mechanism, however, appears to operate only in the presence of serum, as after prolonged growth with NGF in serum-free medium, cells have a low reductase activity.5. PC-12 cells grown in the absence of NGF are highly sensitive to lovastatin (25 µM) and more than 70% of the cells die after 48 hr. NGF confers lovastatin resistance on cells grown in the presence or in the absence of serum (only 30–40% cell death after 48 hr with lovastatin).6. NGF-induced resistance on lovastatin develops with time and is apparent only in the well-differentiated PC-12 cells whether or not the cells express a high reductase activity.7. Thus, levels of HMG-CoA reductase activity and lovastatin resistance in PC-12 cells are not directly correlated, though clearly inversed lovastatin cytotoxicity and elevated reductase activities are expressed during the period of cell proliferation.8. These data suggest that fully differentiated neuronal cells may not be affected by prolonged high doses of lovastatin.     

Expression of recombinant human nerve growth factor in Escherichia coli.

Nerve growth factor (NGF) is a neurotrophic factor for basal forebrain cholinergic neurons and may be of benefit in neurodegenerative diseases of humans. A method is described to obtain significant amounts of biologically active recombinant human NGF (rhNGF) in one step. RhNGF was expressed in E. coli and the majority of the protein accumulated in inclusion bodies. It was immunoprecipitated by a serum against mouse NGF. Solubilization of the inclusion bodies was done in 3M guanidine HCl and renaturation was effected by dilution and air oxidation in the presence of 6 microM CuSO4. Recoveries were 10-12 micrograms of rhNGF per ml of bacterial suspension. Its biological activity was tested in a bioassay system employing sympathetic chick embryo ganglia and was inhibited by the monoclonal antibody 27/21 against mouse NGF.     

Nerve growth factor and serum differentially regulate development of the embryonic otic vesicle and cochleovestibular ganglion in vitro

The preceding paper (P. Bernd and J. Represa, 1989, Dev. Biol. 134) describes the characterization and localization of nerve growth factor (NGF) receptors in inner ear primordia, the otic vesicle (OV) and cochleovestibular ganglion (CVG), obtained from 72-hr (stage 19-20) quail embryos. The studies described in this paper investigated whether NGF serves as a mitogen, a survival factor, and/or a differentiation factor in this system. Explants of isolated OV and CVG were maintained for 24 hr in serum-free medium alone (M-199), M-199 containing serum, M-199 containing NGF, or M-199 containing both serum and NGF. [3H]Thymidine was also present for the entire culture period. Both OV and CVG incorporated greater amounts of [3H]thymidine in the presence of serum or NGF, and their combined effect was additive. NGF's effects were dose dependent, saturable, and specific (blocked by anti-NGF). NGF caused little or no morphological differentiation of OV and no increase in protein levels, in contrast to OV grown in the presence of serum. CVG had both cochlear and vestibular portions present in all cases, but the apparent size and protein content of CVG was increased in the presence of either serum or NGF. Effects of serum and NGF were completely, but reversibly, blocked by amiloride, suggesting that the Na+-H+ exchange system had been activated. In order to determine whether increases in [3H]thymidine incorporation were due to increased cell survival or perhaps to an increase in proliferation, explants were initially grown for a 24-hr period in serum-free medium, followed by reactivation for an additional 24 hr in medium containing serum and/or NGF. It is likely that cells requiring either serum or NGF for survival would die during a 24-hr period in their absence. Our results revealed that the level of [3H]thymidine incorporation in OV was the same after reactivation. In the case of CVG, only NGF treatment yielded similar results; [3H]thymidine incorporation was lower in CVG reactivated with serum. It appears, therefore, that serum has probable proliferative effects upon OV and CVG, as well as survival effects for CVG. NGF, however, does not appear to affect survival in either OV or CVG, so that increases in [3H]thymidine incorporation in response to NGF are most likely due to proliferative effects upon OV or CVG, at least at this embryonic stage.     

Factors in the rat submaxillary gland that stimulate growth of cultured glioma cells: identification and partial characterization

The effect of rat submaxillary extract on the growth of rat C6 glioma cells in serum-free culture has been examined. Extracts (10-15 microgram/ml) of submaxillary glands from both male and female rats markedly enhanced the growth of serum-deprived C6 cells and, in combination with insulin, transferrin, and NIH-LH (a source of fibroblast growth factor), were able to stimulate C6 cell growth to an extent comparable to that achieved with an optimal amount of fetal calf serum. The mitogenic activity of rat submaxillary extracts was found to be heat-labile, acid-stable, and partially inactivated by protease and 2-mercaptoethanol. Under our assay conditions, biologically active preparations of purified mouse submaxillary gland epidermal growth factor (EGF) or nerve growth factor (NGF) were not mitogenic for C6 cells, nor was the mitogenic activity of rat submaxillary extracts inhibited by antiserum to these mouse submaxillary gland growth factors. These results suggest that the active component(s) of rat submaxillary extracts is unrelated to either EGF or NGF. The growth-enhancing effect also appears unrelated to esteropeptidase activity present in these extracts since the mitogenic activity was unaffected by several protease inhibitors. Moreover, two purified mouse submaxillary gland arginylesteropeptidases, EGF-binding protein and gamma-subunit of 7 S NGF, were unable to elicit a comparable growth response even when added to cell culture medium at unreasonably high concentrations. The C6 cell mitogenic activity of crude submaxillary extracts could be separated into two biologically similar components by either gel filtration on Sephadex G-100, preparative isoelectric focusing in a pH gradient of 3-10, or adsorption to DEAE-cellulose followed by elution with a sodium chloride gradient. One of the active components was acidic in nature and had an apparent molecular weight of 40,000, while the other was near neutral in charge and possessed a molecular weight of approximately 20,000. The relationship between these two C6 cell mitogenic components and the rat submaxillary gland component responsible for stimulating Balb/c-3T3 cell growth in serum-free, factor supplemented medium (McClure et al., 1979, J. Cell Biol. 83:96a) is also discussed.     

Cell cycle-specific action of nerve growth factor in PC12 cells: differentiation without proliferation.

PC12 cells were manipulated in such a way as to permit the study of differentiation-specific responses independently from proliferative responses. Cells were starved for serum then exposed to nerve growth factor (NGF) or serum. Following addition of serum, cells incorporated thymidine in a synchronous manner. Subsequent to the wave of DNA synthesis, the cell number increased approximately two-fold. Addition of NGF to serum-starved cultures had no measurable effect on either parameter. Neurite outgrowth was more rapid and extensive and appearance of Na+ channels, measured as saxitoxin binding sites, more rapid than when NGF was added to exponentially-growing cells. Epidermal growth factor receptors were heterologously down-regulated by NGF with similar kinetics under both conditions. Induction of the proto-oncogene c-fos by NGF was also greater in the serum-starved cells than in exponentially-growing cultures. These results indicated that serum starvation resulted in synchronisation of the cultures and that NGF action may be cell cycle-specific. Analysis of the cellular response to NGF at different times during the cell cycle showed that c-fos was induced in the G1 phase but not in S or G2. Fluorescence-activated cell sorter analysis demonstrated that addition of NGF to exponentially-growing cells, resulted in their accumulation in a G1-like state. With regard to the study of the mechanism of NGF action, these results illustrate that measurements of NGF effects on specific components in the signal transduction pathway may be confounded by the use of exponentially-growing cultures.     

Nerve Growth Factor Protects PC12 Cells Against Peroxynitrite-Induced Apoptosis via a Mechanism Dependent on Phosphatidylinositol 3-Kinase

Abstract: Nerve growth factor (NGF) prevents apoptosis induced by the oxidant peroxynitrite in undifferentiated PC12 rat pheochromocytoma cells. Previous studies have shown that activation of phosphatidylinositol 3-kinase (PI 3-kinase) by NGF via the TrkA receptor tyrosine kinase protects PC12 cells from serum deprivation-induced apoptosis. We found that two PI 3-kinase inhibitors, wortmannin and LY294002, eliminated the protection NGF provided against peroxynitrite-induced apoptosis at concentrations consistent with their effectiveness as PI 3-kinase inhibitors. When the activity of PI 3-kinase was assayed in phosphotyrosine immunoprecipitates after treatment of PC12 cells with peroxynitrite, PI 3-kinase activity was reduced by 50% of that detected in control cells, whereas PI 3-kinase activity in NGF-treated cells was unaffected by peroxynitrite. If an antibody against PI 3-kinase was used to immunoprecipitate the enzyme, treatment with peroxynitrite had no effect on activity. Therefore, peroxynitrite appeared to disrupt interactions between PI 3-kinase and phosphotyrosine proteins, rather than directly inhibiting the enzyme. NGF also activates p21^Ras-dependent pathways, but this did not appear to be required for NGF to exert its protective effect against peroxynitrite. PC12 cells expressing a dominant inhibitory mutant of p21^Ras were equally susceptible to peroxynitrite-induced apoptosis, which was prevented by NGF. Wortmannin was also able to block the protective effect of NGF in the p21^Ras mutant cell line. Although many signaling pathways are activated by NGF, these results suggest that a PI 3-kinase-dependent pathway is important for inhibiting peroxynitrite-induced apoptosis.     

Putrescine-Modified Nerve Growth Factor: Bioactivity, Plasma Pharmacokinetics, Blood-Brain/Nerve Barrier Permeability, and Nervous System Biodistribution

Abstract: Previous investigations from our laboratory have demonstrated that the covalent modification of a variety of proteins, including antioxidant enzymes, with the naturally occurring polyamines—putrescine (PUT), spermidine, and spermine—dramatically increases their permeability coefficient-surface area product (PS) at the blood-brain and blood-nerve barriers after parenteral administration. In the present study, we have covalently modified nerve growth factor (NGF) with PUT by targeting carboxylic groups for their graded modification by controlling the ionization of these groups with pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western, and isoelectric focusing analyses demonstrated conversion of NGF to its polyamine-modified derivatives at different pH values. Although the immunoreactivity of PUT-NGF determined by ELISA and western analysis decreased with decreasing pH, the biological activity of PUT-NGF was not affected at any pH as determined by survival and neurite extension of dorsal root ganglia and PC12 cultures. Plasma pharmacokinetics after a single intravenous bolus administration revealed intact PUT-NGF through 10 min and 73–82% intact protein at 15 min. The PS value for PUT-NGF was maximized and the residual plasma volume (V_p) of the protein in the blood vessels minimized when the pH of the modification reaction was >6.4. The biodistribution of PUT-NGF at 15 min showed 22–33% intact protein in different brain regions, which represented 0.4–5.9 ng of PUT-NGF in different brain regions, a physiological dose that is capable of eliciting a bioresponse. The design of this polyamine-modified NGF derivative that has enhanced permeability at the blood-brain and blood-nerve barriers with retained bioactivity may obviate the necessity to create small-molecule mimics of NGF and may be applicable to neurotrophins, engineered multifunctional chimeric neurotrophins, antioxidant enzymes, and other therapeutic proteins with specific clinical application to neurological diseases.     

Nerve growth factor in human amniotic and cerebrospinal fluid

Nerve growth factor (NGF) is a polypeptide hormone involved in development of the sympathetic and central nervous systems. The detection and measurement of NGF in clinical samples would be useful in evaluating its role in various disease states. In this report, NGF activity and protein levels have been investigated in human amniotic fluid and cerebrospinal fluid samples. In amniotic fluid, NGF activity was found at levels ranging from less than 10 pM to nanomolar. The activity in all samples was blocked by polyclonal and monoclonal antibodies to mouse NGF. The finding of NGF in clinically obtainable samples raises the possibility of correlating NGF levels with a variety of disorders in which changes in NGF levels or activity have been implicated.     

On the relationship of zinc ion to the structure and function of the 7S nerve growth factor protein.

The 7S nerve growth factor (7S NGF) is an oligomeric protein consisting of three distinct classes of subunits, alpha,beta, and gamma (A. P. Smith, S. Varon, and E. M. Shooter (1968), Biochemistry 7, 3259). The beta subunit contains the growth promoting activity while gamma is a potent esteropeptidase. The proteolytic activity of gamma is virtually completely inhibited in the 7S NGF aggregate (L. A. Greene, E. M. Shooter, and S. Varon (1969), Biochemistry 8, 3735). In this paper, we report that divalent metal ion chelating agents effect a seven- to tenfold increase in the esteropeptidase activity of 7S NGF at pH 7.40. Plots of esteropeptidase activity vs. chelator concentration give saturation curves which are either sigmoidal (EDTA) or hyperbolic (o-phenanthroline) depending on the chemical structure of the chelator. A survey of common divalent metal ions shows that only zinc ion (Ki = 8 times 10(7) M) and, to a lesser extent, cadmium ion are effective, reversible inhibitors of both 7S NGF and the gamma subunit esteropeptidase activities. We have found that during isolation of 7S NGF, Zn2+ is selectively associated with the oligomer in a ratio of approximately 1-2 g-atoms of zinc/mol of 7S NGF with an apparent affinity which is orders of magnitude tighter than is indicated by the Ki value for the gamma subunit. Dialysis to pH 4.0 where 7S NGF is known to undergo a reversible dissociation (A. P. Smith, S. Varon, and E. M. Shooter (1968), Biochemistry 7, 3259) brings about a tenfold reduction in the zinc ion content of the protein. This reduction is reversed on dialysis back to pH 7.4. In contrast, the isolated subunits contain only trace amounts of zinc ion at pH 7.4. Preliminary metal ion exchange experiments indicate that, of the common metal ions known to substitute for zinc in other zinc-metalloproteins, only cadmium ion is effective in substituting for zinc ion in 7S NGF. The fact that zinc ion is specifically bound to native 7S NGF, and that the zinc ion content of the system is critically dependent on the subunit aggregation state strongly suggests that zinc ion is an integral structural component of native 7S NGF.     

等电聚焦法测定2.5S NGF的等电点

采用水平等电聚焦法测定了２５Ｓｍ神经生长因子（ＮＧＦ）的等电点。经测定,纯化的２５ＳｍＮＧＦ的等电聚焦为三条带,这与文献报道相一致,等电点ｐＨ值分别为ｐＨ８７、９０、９３,其主要成分为β亚基ＮＧＦ及其修饰物的混合物,包括在羧基端失去８个氨基酸残基和在氨基端失去１个精氨酸残基,由这三种组分构成的ＮＧＦ统称为２５ＳｍＮＧＦ。