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.     

Dietary regulation of levels of active mRNA coding for amylase and serine protease zymogens in the rat pancreas

Translation products of a reticulocyte lysate reaction, programmed with poly(A)-rich RNAs from the male mouse submaxillary gland, were subjected to affinity chromatography on a tubulin-Sepharose column. Analysis of the bound proteins in sodium dodecylsulfate/polyacrylamide gels revealed two polypeptides of Mr 27 000 and 45 000, that were shown to bind to tubulin in a specific manner. These polypeptides were absent from the translation products coded by poly(A)-rich RNAs from the female mouse. They were eluted from the tubulin-Sepharose resin under conditions similar to those employed for the dissociation of immune complexes. The Mr-27 000 and Mr-45 000 proteins were identified by immunoprecipitation with specific antisera as the precursors of the gamma subunit of the nerve growth factor (NGF) and renin respectively. These two precursors as well as a third, unidentified polypeptide of Mr 38 000, probably unrelated to the beta subunit of NGF, bound also to microtubules. The mature form of renin, purified from the submaximillary gland, also displayed an affinity for the microtubules. In contrast, the mature form of the gamma subunit of NGF did not bind to the microtubules. The possible involvement of the microtubules (tubulin) in the biosynthesis of these two secretory proteins is discussed.     

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.     

Level of the nerve growth factor activity in the submaxillary glands of genetically dystrophic mouse (C57BL/6J)

To examine the relationship between NGF activity and neurologic disorders, the level of NGF activity was studied in the submaxillary glands of both normal and dystrophic mice. The level of NGF activity in the mice was remarkably lower than that observed in normal mice. The data suggest that NGF activity may have some relation to the cause of the disease.     

Circular dichroism and crosslinking studies of the interaction between four neurotrophins and the extracellular domain of the low-affinity neurotrophin receptor.

Interactions between the purified recombinant receptor extracellular domain (RED) of the human low-affinity neurotrophin receptor (LANR) and recombinant human brain-derived neurotrophic factor, neurotrophin-3 (NT-3) and neuotrophin-4/5 have been studied by chemical crosslinking and circular dichroism. Conformational changes subsequent to binding have been shown by these procedures. First, relative affinities of the neurotrophins for RED were determined by binding competition assays in which radioiodinated nerve growth factor (NGF) from mouse submaxillary gland was crosslinked to RED in the presence of varying amounts of unlabeled neurotrophin competitors. RED bound each of the 3 recombinant human neurotrophins with affinities that were indistinguishable from authentic mouse NGF. These results are the first measurement of binding of the neurotrophin family to their common receptor using purified components. In order to study the effect of binding on the conformation of the proteins, CD measurements were made before and after mixing neurotrophins and RED, as had previously been done with NGF and RED (Timm DE, Vissavajjhala P, Ross AH, Neet KE, 1992, Protein Sci 1:1023-1031). Similar changes in CD spectra occurred upon combination of each of the neurotrophins and RED, with negative changes near 220-225 nm and positive changes near 190-200 nm; however, significant differences existed among the various neurotrophin-RED difference spectra. The NT-3/RED complex showed the largest spectral change and NGF the smallest. Thus, specific conformational changes in secondary structure of neurotrophin, RED, or both accompany the binding of each neurotrophin to the extracellular domain of the LANR.     

Retrograde Transport of Endogenous Nerve Growth Factor in Superior Cervical Ganglion of Adult Rats

The concentration of naturally synthesized nerve growth factor (NGF) was measured in various tissues of adult rats, using a highly sensitive two-site enzyme immunoassay. The highest concentration was found in the superior cervical sympathetic ganglion (SCG). Transection of the postganglionic external carotid nerve (ECN) reduced the ganglionic level of NGF more than did section of the internal carotid nerve (ICN). When both the preganglionic nerve and the ECN were cut, the ganglionic NGF level decreased even more. On the other hand, when the preganglionic nerve and the ICN were both sectioned, leaving the ECN intact, endogenous NGF content in the SCG was significantly enhanced 3-9 h after operation. Bilateral extirpation of submaxillary gland produced a rapid decrease in ganglionic NGF 3-6 h after operation, and even unilateral removal of one salivary gland caused a decrease in both ganglia, which was however much greater in the ipsi- than in the contralateral ganglion. Removal of the eyeballs caused a much smaller reduction in ganglionic NGF than did removal of the glands. These results suggest that the endogenous NGF that accumulates in the SCG is mostly synthesized in the submaxillary gland rather than in the iris, and that it is transported to the SCG, mostly via the ipsilateral ECN.     

A Highly Sensitive Enzyme Immunoassay for Mouse β Nerve Growth Factor

Abstract: A sensitive two-site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti-mouse β NGF antibody IgG coated to a polystyrene tube and anti-mouse β NGF antibody Fab'-linked β- d -galactosidase (β- d -galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two-site radioimmunoassay; (b) antibody Fab'-β- d -galactosidase complex is more stable than ¹²⁵I-labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice.     

Effect of mouse submaxillary gland and NGF preparations on competent chick ectodermin vitro,studied by the immunofluorescence method

Summary The submaxillary gland of the male mouse, but not the female, was shown to act strongly neuralizing on competent chick ectoderm, recorded as frequency of neural antigen producing cultures. NGF preparations from mouse submaxillary gland were shown to cause a high frequency of neural antigen producing cultures. The effect of the NGF preparations diminished with time and ceased after storage at –90° C for 17 days. The possible role of NGF in the present system is discussed; but the possibility could not be excluded that factors other than NGF present in the submaxillary gland and the NGF preparations cause the competent ectoderm to neuralize.     

Prostaglandins and renin release from submaxillary glands in vitro

The present studies were undertaken to investigate the effect of prostaglandins (PGs) on renin release from the submaxillary glands of mice. Pooled mouse submaxillary gland slices were incubated in Krebs-Henseleit buffer solution following a preincubation period, and renin release was measured by a radioimmunoassay for the direct measurement of submaxillary gland renin. Arachidonic acid (AA) significantly stimulated renin release at 10, 20, and 30 min of incubation. These increases of renin release were abolished by the presence of indomethacin. The synthetic prostaglandin endoperoxide analogue (EPA) strongly stimulated renin release at 10, 20, and 30 min of incubation. However, at a higher concentration the stimulating effect of EPA virtually disappeared. PGI2 caused the highest increase of renin release at 10 and 20 min of incubation. At higher concentrations the effect of PGI2 on renin release was drastically reduced, although it was still statistically significant. PGE2 and PGF2 alpha also exerted a significant increase in renin release; however, the extent of this effect was much less than that of EPA and PGI2. Other prostaglandins such as PGE1, PGA2, PGD2, PGF1 alpha, and 6-keto-PGF1 alpha were found to have no significant effect on renin release. These results suggest that the prostaglandin system directly affects renin release from submaxillary gland independent of systemic hemodynamic and neurogenic influences.     

Enhancement of angiogenic effect of co-transfection human NGF and VEGF genes in rat bone marrow mesenchymal stem cells

The current study explored the feasibility and efficacy of co-transfection of the human nerve growth factor (NGF) and vascular endothelial growth factor 165 (VEGF165) genes in rat bone marrow mesenchymal stem cells (BMSCs). The obtained hNGF and vascular endothelial growth factor (VEGF) cDNAs were cloned into the pEGFP-C1 expression vector to construct the recombinant vectors. Co-transfection in rat BMSCs was performed and the expressions of both genes were detected by RT-PCR, Western blot, and enzyme-linked immunospecific assay. The biological activity of recombinant NGF and VEGF proteins was confirmed using the Chick Chorioallantoic Membrane (CAM) assay. NGF and VEGF genes could be expressed successfully in rat BMSCs. The recombinant NGF and VEGF from the rat BMSCs showed a more significant synergetic biological activity compared with single recombinant NGF or VEGF. These findings demonstrate that the co-transfection of hNGF + VEGF genes can enhance the angiogenic effect in vivo.     

Characterization of primary translation products from ovine and rat salivary gland mRNAs

Ovine and rat salivary gland mRNAs have been prepared and their translation products characterized. A 60 kD translation product from ovine submaxillary and sublingual gland mRNAs is identical in mass to the ovine apomucin. Two additional ovine translation products, 25 and 40 kD, are specific to mucin-producing salivary glands. Four rat mRNA translation products are encoded by mucin-producing salivary glands (38, 44, 67, 69 kD). These polypeptides were not detected in the parotid gland mRNAs, a serous gland. Each of these products has a high level of [3H]serine incorporation, a characteristic of mucins. The nature of these products suggests that they are mucins or mucin-like and that their molecular weights should approximate that of the corresponding apomucins.     

Structure and expression of the nerve growth factor gene in Xenopus oocytes and embryos

A large part of the coding portion of the Xenopus nerve growth factor (NGF) gene has been identified and cloned by the use of a chicken cDNA probe and its sequence has been determined. Comparison of the derived amino acid sequence of mature Xenopus NGF with that of other species showed a high conservation, whereas comparison of the prepropeptide showed large divergent regions alternated with short conserved regions. Expression of the NGF gene was examined during development of oocytes and embryos. Surprisingly, NGF mRNA was found in the oocyte; it is present in small previtellogenic as well as in fully grown oocytes. NGF mRNA, passed to the embryo at fertilization, is degraded before the gastrula stage and starts accumulating again around the stage of the neurula. The association of NGF mRNA with polysomes is indicative of NGF synthesis during oogenesis. In fact, by using antibodies against mouse NGF it was possible to reveal NGF molecules present as precursors. These molecules accumulate during oogenesis and are maintained in the embryos up to the blastula stage; a very faint band corresponding to a smaller size peptide is sometimes detected. A maternal role for the NGF can be proposed, although a possible activity of NGF in the oocyte cannot be ruled out.     

Optimedin induces expression of N-cadherin and stimulates aggregation of NGF-stimulated PC12 cells

Optimedin, also known as olfactomedin 3, belongs to a family of olfactomedin domain-containing proteins. It is expressed in neural tissues and Pax6 is involved in the regulation of its promoter. To study possible effects of optimedin on the differentiation of neural cells, we produced stably transfected PC12 cell lines expressing optimedin under a tetracycline-inducible promoter. Cells expressing high levels of optimedin showed higher growth rates and stronger adhesion to the collagen extracellular matrix as compared with control PC12 cells. After stimulation with nerve growth factor (NGF), optimedin-expressing cells demonstrated elevated levels of N-cadherin, beta-catenin, alpha-catenin and occludin as compared with stimulated, control PC12 cells. Expression of optimedin induced Ca(2+)-dependent aggregation of NGF-stimulated PC12 cells and this aggregation was blocked by the expression of N-cadherin siRNA. Expression of optimedin also changed the organization of the actin cytoskeleton and inhibited neurite outgrowth in NGF-stimulated PC12 cells. We suggest that expression of optimedin stimulates the formation of adherent and tight junctions on the cell surface and this may play an important role in the differentiation of the brain and retina through the modulation of cytoskeleton organization, cell-cell adhesion and migration.     

Nerve growth factor-mediated inhibition of apoptosis post-caspase activation is due to removal of active caspase-3 in a lysosome-dependent manner

Nerve growth factor (NGF) is well characterised as an important pro-survival factor in neuronal cells that can inhibit apoptotic cell death upstream of mitochondrial outer membrane permeabilisation. Here we addressed the question of whether NGF can also protect against apoptosis downstream of caspase activation. NGF treatment promoted a rapid reduction in the level of the p17 subunit of active caspase-3 in PC12 cells that had been induced to undergo apoptosis by various cytotoxins. The mechanism involved TrkA-dependent activation of extracellular signal-regulated kinase (ERK1/2) but not phosphatidylinositol 3-kinase (PI3K)/Akt, and de novo protein synthesis. Involvement of inhibitor of apoptosis proteins (IAPs) and proteasomal degradation were ruled out. In contrast, inhibition of lysosome function using chloroquine and concanamycin A reversed NGF-induced removal of p17. Moreover, in NGF-treated cells, active caspases were found to be localised to lysosomes. The involvement of macroautophagy and chaperone-mediated autophagy were ruled out. Taken together, these findings suggest an anti-apoptotic mechanism by which NGF induces removal of active caspase-3 in a lysosome-dependent manner.     

蛇毒神经生长因子生物活性研究

从蛇毒中分离的神经生长因子（ＮＧＦ）,经用鸡胚背根神经节做生物活性测定,发现能促进神经节树突最大生长的最小ＮＧＦ浓度为２．５ｎｇ／ｍｌ。半成品的毫克蛋白（ｍｇＰ）比活性在１．５４×１０５～５．３５×１０５Ｕ／ｍｇＰ之间。经用ＨＰＬＣ和ＳＤＳ－ＰＡＧＥ电泳做纯度分析,主峰面积在９５％以上。并从四种ＮＧＦ成品赋形剂中选出了理想的配方     

雌二醇对大鼠颌下腺EGF、NGF生成的影响

为了研究雌二醇（estradiol-17β,E2）对颌下腺表皮生长因子（epidermal growth factor,EGF)和神经生长因子（nerve growth factor,NGF)生成的影响,研究采用免疫组织化学方法,对外源性投予雌二醇后的大鼠颌下腺进行了观察。结果证实E2明显促进颌下腺EGF和NGF的生成。提示雌二醇可能对EGF和NGF的合成起重要的调节作用。     

Renin in the mouse submaxillary gland has a molecular weight of 40,000.

The availability of pure submaximillary renin, its antibody and pure specific immunoreactive Fab fragments of the antirenin molecule were used in an attempt to detect in which form renin is stored in the submaxillary gland. The proteolytic activity of serine-, metallo- and sulfhydryl enzymes during homogenisation was inhibited, but no inactive or high molecular weight form could be detected enzymatically or antigenically after gelfiltration. Nor were they demonstrable in crossed immuno-electrophoresis by using antibodies elicited against pure renin. Furthermore, pepstatin which additionally inhibits acid proteases, including a possible autoactivation of renin, and renin specific Fab fragments, were added, the latter in order to steric hinder proteolytic attack on a possible renin precursor. The renin-Fab complex was purified by precipitation with anti-Fab antibodies. No high molecular weight renin was demonstrable in SDS polyacrylamide gel electrophoresis. The only form of renin demonstrable in the submaxillary gland of mice was the fully active 40,000 dalton form. Its specific enzymatic activity was identical to that of pure submaxillary renin, being 0.4 . 10(-3) Goldblatt unit . ng-1.     

Nerve growth factor stimulates regeneration of perivascular nerve,and induces the maturation of microvessels around the injured artery

An immature vasa vasorum in the adventitia of arteries has been implicated in induction of the formation of unstable atherosclerotic plaques. Normalization/maturation of the vasa vasorum may be an attractive therapeutic approach for arteriosclerotic diseases. Nerve growth factor (NGF) is a pleotropic molecule with angiogenic activity in addition to neural growth effects. However, whether NGF affects the formation of microvessels in addition to innervation during pathological angiogenesis is unclear. In the present study, we show a new role for NGF in neovessels around injured arterial walls using a novel in vivo angiogenesis assay.     

L cells potentiate the effect of the extracellular NGF activity in co-cultures with PC12 pheochromocytoma cells

Two mouse cell lines, 3T3 and L, reported to secrete an NGF-like activity in the culture medium were co-cultured with the pheochromocytoma cell line PC12 which responds to NGF in vitro. In these co-cultures mitomycin-treated L or 3T3 cells were employed at low cell density (1 000 cells/cm2). L cells, but not 3T3, promoted efficiently neurite outgrowth of PC12. The response of the PC12 cells was blocked by an antiserum to male mouse submaxillary gland beta NGF. The NGF secreted by the L cells and immunoprecipitated by this antiserum co-migrated with the submaxillary gland beta NGF monomer in SDS-polyacrylamide gels. Surprisingly the neurite-promoting activity of media conditioned by L or by L-PC12 co-cultures was at most one-tenth of that expected on the basis of the response of PC12 cells in the co-cultures. This was not due to proteolytic degradation of the NGF-like factor or to losses by manipulation of the media. It seems therefore that co-cultures provide conditions which enhance the effect of the factor. Possible mechanisms responsible for this effect are discussed.