De novo synthesis of NGF subunits in S-180 mouse sarcoma cell line

It is an accepted hypothesis that the nerve growth factor protein (NGF) plays an important role in the development of vertebrate sympathetic and sensory ganglia and has effects on some central neurons. The best known NGF species is that isolated from the mouse submaxillary gland, MSG-NGF. MSG-NGF can be isolated as a subunit containing protein, 7S-NGF, made up of three dissimilar subunits called alpha-, beta-, and gamma-NGF. Beta-NGF is the biologically active subunit and its synthesis in vivo and in vitro has been demonstrated. Less is known about the synthesis of the alpha- and gamma-NGF or the assembly of the subunits into the 7S complex. In order to develop a clonal model system for the study of NGF synthesis, processing and secretion, affinity chromatography techniques were applied to cell extracts of S180 mouse sarcoma, a cell line known to synthesize NGF. After incubating S180 cells in³⁵S-Methionine, cell extracts were exposed to antibody directed against alpha-NGF, gamma-NGF or beta-NGF covalently bound to Sepharose beads in order to elute and characterize the desired NGF subunits. Parallel experiments using immunoabsorbed [³⁵S]Methionine-beta-NGF were carried out in the presence or absence of excess NGF, in order to demonstrate the specificity of this procedure. Affinity chromatography with a substrate analogue to arginine ester bound to Sepharose beads was also used to isolate de novo synthesized gamma-NGF. We were able to show that the S180 line synthesized alpha-, beta-, and gamma-NGF indistiguishable from alpha-, beta-, and gamma-NGF isolated from mouse submaxillary gland in terms of antigenic and physicochemical properties, and biological and enzymatic activities. These results are consistent with the hypothesis that NGF is synthesized, assembled and secreted by a single cell type.Special Issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

Zinc-containing 7S-NGF complex. Evidence from zinc histochemistry for localization in salivary secretory granules

7S-NGF is a pro-protein containing a neurotrophic subunit, beta-NGF, which has been localized by immunocytochemistry to the granules of granular convoluted tubule (GCT) cells in certain murine salivary glands [Watson et al., Anat Rec (1985) 213:365]. The 7S-NGF pro-protein contains zinc and is stabilized by zinc ions [Pattison and Dunn, Biochemistry (1976) 15:3696]. In the present work, dithizone, toluene sulfonamide quinoline (TSQ), and neo-Timm's methods for zinc were used to determine whether zinc histochemistry could be used to visualize the zinc associated with the 7S-NGF complex and, if so, whether zinc histochemistry might corroborate the reported localization of the 7S-NGF complex in GCT secretory granules. The results indicate that intensity of zinc staining varies with the reported variations in NGF levels in different salivary glands, and that the zinc is selectively concentrated in the GCT secretory granules. We suggest that zinc histochemistry may be a useful marker for the presence of the zinc-stabilized 7S-NGF pro-protein.     

Immunocytochemical localization and concentrations of the α and γ subunits of 7S-nerve growth factor in the submandibular gland of the mouse

Summary For unexplained reasons, nerve growth factor (NGF) exists in very high concentrations in the submandibular gland of the mouse. The NGF in the gland, called 7S-NGF, is a non-covalent complex of three protein subunits, named -, - and -NGF. All the known biological activity resides in the -NGF subunit, and previous studies have shown that -NGF is present in much greater concentrations in the male submandibular gland than in the female gland. The higher concentration in the male is due to the fact that -NGF is synthesized in the granular tubule cells of the submandibular gland. These cells are much more numerous in the male gland.In contrast to -NGF, neither the concentrations of and subunits nor their cellular localization in the mouse submandibular gland have been established. In this study, radioimmunoassays specific for . and subunits determined that both are present in much higher concentrations in the male gland. Immunocytochemical work localized both subunits in the granular tubule cell in the male and female submandibular gland. This indicates that all the components of 7S-NGF exist in a single cell type in the gland and suggests that 7S-NGF can be formed within this cell and secreted as a complex into the saliva.     

Absence of the alpha and gamma subunits of 7S nerve growth factor in denervated rodent iris: immunocytochemical studies

Immunocytochemical studies were performed to determine if denervated rodent iris produces nerve growth factor (NGF) in a form chemically similar to that of the 7S NGF complex in mouse submandibular glands. Antisera to the alpha, beta, and gamma subunits of 7S NGF were raised in rabbits and characterized on immunoblots of SDS-containing polyacrylamide gels. Antisera were applied to stretch preparations of rat and mouse irides that were cultured for periods of 2 to 6 days or sympathetically denervated by superior cervical ganglionectomy and left in situ 4 days. Antibody binding was visualized by indirect immunofluorescence. In control studies done on plastic sections of mouse submandibular glands, antisera co-localized the three subunits of 7S NGF within secretory granules of granular tubule cells. In denervated rat iris, beta NGF immunoreactivity was evident in a cellular plexus that resembled in distribution and morphology nerve fibers in the normal iris, in agreement with a previous study (R.A. Rush (1984). Nature (London) 312, 364-367). Identical staining patterns were observed in mouse iris. In neither rat or mouse, however, did the nerve-like processes stain with antibodies suggests that the NGF-like protein in denervated rodent iris is not synthesized as part of the 7S NGF complex. Iris also did not react with antibodies to epidermal growth factor, a protein co-localized with NGF in mouse submandibular glands and in guinea pig prostate.     

Processing of the native nerve growth factor precursor to form biologically active nerve growth factor

In the mouse submaxillary gland beta nerve growth factor (beta-NGF) forms a complex with two members of the kallikrein family of serine proteases, termed the alpha- and gamma-subunits of NGF. We demonstrate that the beta-NGF precursor produced in mammalian cells via a recombinant vaccinia virus can be cleaved by stoichiometric quantities of the gamma-subunit to produce beta-NGF. Trypsin in catalytic quantities also produces native beta-NGF. Proper cleavage depends critically on the conformation of the precursor. beta-NGF has at least 10-fold more biological activity than its precursor.     

Developmental changes of nerve growth factor levels in sympathetic ganglia and their target organs

The predominant source of nerve growth factor (NGF) used by mature sympathetic neurons originates in their target organs (Heumann, R., Korsching, S., Scott, J., and Thoenen, H. (1984), EMBO J. 3, 3183-3189; Korsching, S., and Thoenen, H. (1985), J. Neurosci. 5, 1058-1061). We have determined the NGF content of two sympathetically innervated mouse organs, submandibular gland and heart ventricle, and of sympathetic ganglia from mouse and rat between embryonic Day 12 (E12) and adulthood. NGF levels were measured by a two-site enzyme immunassay (Korsching, S., and Thoenen, H. (1983), Proc. Natl. Acad. Sci. USA 80, 3513-3516). In heart ventricle and submandibular gland, NGF first became detectable around the time of initial innervation by sympathetic neurons (E12 and E13, respectively) and increased respectively 14- and 7-fold in the following 2 days, to reach adult levels already at E14 for heart ventricle (1.4 +/- 0.2 ng NGF/g wet wt). NGF in the superior cervical ganglion (SCG) was first detected at the same time as in its target organ, the submandibular gland. NGF content in the SCG then increased 6-fold during the next 2 days and continued to increase until the end of the third postnatal week, when adult levels were reached. Although the levels of NGF in the adult mouse submandibular gland are sexually dimorphic and six orders of magnitude higher than those in other sympathetic target organs, no sex difference in the NGF content was found in either developing submandibular gland or SCG until the end of the third postnatal week. Moreover, the steep NGF increase observed in the male submandibular gland after postnatal Day 18 (250-fold within the following 3 days and up to the 55,000-fold in the next 7 days) was not reflected in a corresponding increase in the NGF content of the male SCG. These data indicate that, in accordance with earlier findings (see Levi-Montalcini, R., and Angeletti, P. U. (1968), Physiol. Rev. 48, 534-569), SCG neurons do not have access to the large amounts of NGF synthesized during and after adolescence in the mouse submandibular gland. Our results support the concept that initial fiber outgrowth of sympathetic neurons is neither dependent on NGF nor mediated by it. The time course of NGF levels in the SCG is consistent with the concept that sympathetic neurons are provided with NGF by means of retrograde axonal transport from the innervated organs already early in development.     

Catecholamines induce an increase in nerve growth factor content in the medium of mouse L-M cells

L-M cells, a mouse fibroblast cell line, synthesized and secreted a nerve growth factor (NGF). The neurite outgrowth stimulatory activity, immunoreactivity, molecular weight, and isoelectric point of L-M cell NGF were identical to those of beta-NGF of the mouse submaxillary gland. Treatment of the cells with either norepinephrine or epinephrine in the range of 0.05-0.2 mM for 24 h resulted in a 3-20-fold increase in NGF content in the medium of the L-M cells. The NGF of epinephrine-treated cell was identical to that of control cell. The stimulation of the increase in NGF content was observed after a 4-h lag time. The rate of incorporation of [3H]leucine into trichloroacetic acid-insoluble materials was essentially unchanged during the treatment. These results suggested that norepinephrine and epinephrine stimulated the de novo synthesis and secretion of NGF protein. Evidence is also presented to indicate that the effects of the drugs are due to the catechol part of the molecule and not mediated by adrenergic receptors.     

Comparison of 7S nerve growth factor and nerve growth factor I from mouse submandibular glands

7S nerve growth factor (7S NGF) and nerve growth factor I (NGFI) are NGF-containing protein complexes isolated from mouse submandibular glands by different protocols, and reports suggest that the molecules differ chemically. In this study, we compared the molecular properties and subunit compositions of the two proteins. Purified 7S NGF and NGFI electrophoresed to identical positions on polyacrylamide gels in nondissociating buffers, with electrophoretic mobilities indistinguishable from that of unpurified NGF in salivary gland extracts. Ultraviolet absorption curves were identical, and sedimentation coefficients were similar (7.3 +/- 0.25 S for 7S NGF; 7.2 +/- 0.2 S for NGFI) as determined by sedimentation velocity analysis. By sedimentation equilibrium analysis, molecular weights of 135 000-140 000 were obtained for both complexes at protein concentrations in the centrifuge cell greater than 85 micrograms/mL; when protein concentrations within the centrifuge cell ranged from approximately 30 to 100 micrograms/mL at equilibrium, both complexes dissociated. Molecular weight values determined by gel filtration on Bio-Gel P300 and Sephadex G200 resins were similar for both proteins, and the values determined on Sephadex agreed with those obtained by ultracentrifugation. The subunit compositions of the complexes were also similar as determined by nonequilibrium isoelectric focusing, NGFI being composed of proteins that migrated to positions identical with those of the alpha, beta, and gamma subunits of 7S NGF. Furthermore, the stoichiometry of the subunits was similar in the two complexes as determined by radioimmunoassays to each of the subunits and by densitometric analysis of electrophoretic gels. Both methods showed that the complexes contain approximately 2 mol of the alpha and gamma subunits per mole of beta-NGF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential localization of "brain-specific" S-100 and its subunits in rat salivary glands

In the rat, the S-100 antigens in the submandibular gland were found to be immunochemically identical with those in the brain (glial cells) when compared using crossed immunoelectrophoresis. Specific antibodies against the S-100a non-beta and against the S-100 beta subunit were prepared from antibodies against crude S-100 protein and from S-100 components (S-100a and b) by affinity chromatography. In the rat salivary glands a differential distribution of subunit immunoreactivity was clearly evidenced using indirect immunofluorescence. Certain intercalated duct cells of the submandibular gland as well as Schwann cells contained the S-100 beta subunit immunoreactivity exclusively, while other duct cells in parotid, submandibular, and sublingual glands contained S-100a non-beta subunit immunoreactivity. Both subunits were present in astrocytes and ependymal cells. The immunocytochemical localization of alpha and beta subunits is a promising technique for the classification of various types of S-100-containing cells.     

The α and γ subunits of 7S nerve growth factor are present in excess concentrations in the mouse submandibular gland

Summary The 7S nerve growth factor molecule, found in the mouse submandibular gland, is comprised of three distinct protein subunits named , and -NGF. In this paper, radioimmunoassays specific for each subunit were used to measure the concentrations of these subunits in homogenates of mouse submandibular gland. It was determined that there were excess concentrations of both the and subunits, more than enough to bind all of the -NGF in the gland to form 7S-NGF. The radioimmunoassay data was confirmed by gel filtration experiments. In the gel filtration experiments, the excess and subunits eluted at positions which would indicate that these excess subunits were free and not bound in the 7S-NGF complex. The identity of the excess and subunits was substantiated by ion exchange chromatography, isoelectric focusing polyacrylamide gels and immunoblotting experiments. In conclusion, there are considerable quantities of and subunits present in the submandibular gland which are not bound to -NGE The functional significance of these excess concentrations of the and subunits is not known.     

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

Summary Intense labelling of secretory cells in the male mouse submandibular gland was observed afterin 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 ofin 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 ofin situ hybridization and immunocytochemistry on the same section should be particularly useful in cases where NGF is transported away from its site of synthesis.     

Sex-Dependent and Sex-Independent Distribution of the β-Subunit of Nerve Growth Factor in the Central Nervous and Peripheral Tissues of Mice

Levels of the beta-subunit of nerve growth factor (beta-NGF) were measured in the central nervous and peripheral tissues of mice using a highly sensitive, sandwich-type enzyme immunoassay system. Antiserum was raised in rabbits against the 7S form of NGF, which was purified from mouse submandibular glands. beta-NGF-specific antibody isolated on a column of Sepharose CL-4B coupled with purified beta-NGF reacted only with beta-NGF. The assay for beta-NGF was performed by incubation of F(ab')2 fragments of the antibody immobilized on a polystyrene ball with tissue extract and then with the same antibody Fab' fragments labeled with beta-D-galactosidase, followed by measurement of galactosidase activity. Our assay system was found to be highly sensitive (minimal detection limit, 0.3 pg/0.3 ml of assay mixture). Furthermore, the presence of gelatin hydrolysates and protease inhibitors during preparation of tissue extracts enabled us to determine the precise levels of beta-NGF in almost all organs of mice. The amount of beta-NGF in submandibular glands was extremely high, and its level increased rapidly until mice were 2 months of age; then, the level continued to increase slowly until mice were 1 year old (3-5 mg/g of tissue). In serum, some of the 2-month-old males, but none of the females, exhibited a fairly high level of beta-NGF (greater than 100 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical demonstration of epidermal growth factor and nerve growth factor in experimental carcinogenesis in the mouse submandibular gland

Immunohistochemical demonstration of epidermal growth factor (EGF) and nerve growth factor (NGF) was made during chemical carcinogenesis in the mouse submandibular gland. The granular convoluted tubule cells in the normal male submandibular gland contained larger amounts of EGF and NGF than in the female. The initial phase and early stages in chemical carcinogenesis showed degranulation of the granular convoluted tubule cells with a marked decrease in EGF and NGF. Premalignant lesions such as duct-like structures and multicystic lesions showed variable staining for EGF and were usually negative for NGF. Material secreted into the luminal spaces revealed increased staining for EGF and NGF. Scattered tumor cells of the poorly differentiated squamous-cell carcinoma type and desquamated tumor cells contained abundant EGF, but not NGF. No positive reaction for EGF or NGF was found in the induced squamous-cell carcinoma cells.     

Nerve growth factor-like immunoreactivities in rodent salivary glands and testis

Summary A series of polyclonal affinity-purified antibodies against mouse submandibular-gland nerve growth factor (NGF) are described. Using the submandibular gland of the male mouse and indirect immunofluorescence, the specificity and sensitivity of affinity-purified immunoglobulins and various other fractions from the immunized animals have been tested. It will be shown that affinity-purification schemes, including pre-purification of protein A-fractionated immunoglobulins to remove antibodies that bind to unrelated hydrophilic and hydrophobic proteins, significantly enhance the signal-to-noise ratio and specificity of the antibodies. The antibodies effectively detect NGF-like immunoreactivity in both fresh and fixed glandular tissue. Optimal fixation procedures are described. Fluorescence intensities are linearly correlated to log antibody concentration. By use of the best antibody fractions and optimal fixation protocols, the distribution of NGF-like immunoreactivity is described in eight different salivary glands (rat and mouse, male and female, submandibular and sublingual glands). In addition to the well-known large numbers of immunoreactive cells in the submandibular gland of the male mouse, immunoreactive cells were found in the sublingual gland of male mice and in the submandibular and sublingual glands of female mice. One antibody revealed a weak specific fluorescence also in the submandibular gland of the male mouse. In a survey of genital organs of male mice, one antibody revealed fluorescence in the germ cell line. We conclude that several polyclonal affinity-purified antibodies have been characterized that show a strong NGF-dependent binding to the secretory granules of tubular cells in the submandibular gland of male mice. These antibodies should make it possible to locate endogenous and perturbed NGF levels immunocytochemically, e.g., in the peripheral and central nervous system, where NGF concentrations may be several orders of magnitude lower than in the salivary glands.     

Genes for the alpha and gamma subunits of mouse nerve growth factor are contiguous.

Here we describe the structure and linkage of genes encoding the alpha and gamma subunits of mouse nerve growth factor (NGF). These genes are members of the highly homologous glandular kallikrein multigene family. Together with the beta subunit, the alpha and gamma proteins constitute the high mol. wt. (7S) form of NGF isolated from mouse submandibular gland. The gamma subunit is an active serine protease and is thought to cleave pro-beta-NGF to generate the mature growth factor. The alpha subunit has no detectable proteolytic activity, but is essential for the stable formation of 7S NGF. Lack of enzyme activity of the alpha subunit can be attributed, at least in part, to the deletion of 15 nucleotides in a highly conserved coding region which is normally involved in the activation of serine proteases from their inactive zymogen form.     

Nerve growth factor: a chemotactic factor for polymorphonuclear leukocytes in vivo

Previous studies have shown that mouse submandibular gland nerve growth factor (NGF) stimulates chemotactic migration of human polymorphonuclear leukocytes in vitro. The results of the present study demonstrate that subdermal injection of NGF in mice also stimulates rapid and marked chemotactic recruitment of leukocytes. This property of NGF is manifest in the nanomolar range of concentrations, it requires the known serine class protease activity of the growth factor, and it does not require participation of the fifth component of complement. Another, as yet unrecognized, C5-independent pathway must be involved. Chemotactic stimulation of cells involved in the early inflammatory response to injury may help to explain earlier observations that NGF can accelerate the rate of contraction of experimentally induced wounds in mice.     

Secretion of nerve growth factor by central nervous system glioma cells in culture

Bacteriophage immunoassays, radioimmunoassays, and biological assays have been used to measure levels of NGF in media conditioned by rat C-6 glioma cells in culture. By all three criteria, these cells secrete a macromolecule which is indistinguishable from mouse submandibular gland NGF.     

大鼠体内视网膜神经节细胞诱向因子(RGNTF)及其受体的免疫组织化学定位

本文用ＲＧＮＴＦ单克隆抗体及抗独特型单克隆抗体的免疫组织化学反应,对ＲＧＮＴＦ及其受体在 大鼠体内的分布进行了研究．结果显示,大鼠的肾脏、肾上腺、下颌下腺、胃底腺,以及睾丸的生精细胞对 ＲＧＮＴＩＦ均呈现强阳性免疫反应,并对ＲＧＮＴＦ抗独特型单克隆抗体也呈现阳性免疫反应,表明 ＲＧＮＴＦ及其受体有较广泛的分布,这种情况与神经生长因子（ＮＧＦ）及睫状节神经诱向（营养）因子 （ＣＮＴＦ）相类似．但是,ＲＧＮＴＦ及其受体的分布特点和ＮＧＦ、ＣＮＴＦ的分布是不完全相同的,提示作者 分离的ＲＧＮＴＦ与ＮＧＦ和ＣＮＴＦ不是同源物。这样肾上腺皮质、下颌下腺的浆液腺泡及导管上皮细胞、 胃底腺上皮细胞和生精细胞不仅能够产生ＲＧＮＴＦ,也能合成ＲＧＮＴＦ受体。因此,它们对ＲＧＮＴＦ可能 有自分泌的功能,ＲＧＮＴＦ对这些细胞可能有自身调节的效应。     

Nerve growth factor (NGF) is present in human placenta and semen, but undetectable in normal and Paget's disease blood: measurements with an anti-mouse-NGF enzyme immunoassay using a recombinant human NGF reference

An enzyme immunoassay (EIA) originally developed for mouse beta-nerve growth factor (NGF) and commercially available was validated for human NGF. Cell culture medium containing bioactive recombinant human NGF was used as a reference, and mouse 2.5S beta-NGF as a standard. One of three human placentas contained measurable NGF (70 pg/g of tissue of mouse beta-NGF equivalents), a second detectable, and a third undetectable NGF. In three human semen samples NGF content ranged from 0.13 - 1.4 ng/ml. NGF could not be detected in normal human serum and in plasma from patients with Paget's disease, although mouse 2.5S beta-NGF added to human blood could be completely recovered from the serum.