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.     

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.     

Synthetic chimeras of mouse growth factor-associated glandular kallikreins. II. Growth factor binding properties.

Six chimeric constructs of the sequentially similar growth factor-associated kallikreins-epidermal growth factor binding protein (EGF-BP) and the gamma-subunit of nerve growth factor (gamma-NGF)--have been expressed, and their ability to generate complexes with epidermal growth factor (EGF) and beta-NGF, analogous to the high molecular weight forms (7S NGF and HMW-EGF) found in the mouse submaxillary gland, evaluated. The chimeras are distinguished by the interchange of three regions composing the amino, middle, and carboxyl terminal regions that encompass four surface loops possibly involved in specific growth factor interactions. Native beta-NGF (along with native alpha-NGF) formed complexes indistinguishable from naturally occurring 7S NGF, characterized by an alpha 2 beta gamma 2 structure (where beta-NGF is itself a dimer), with recombinant (r) gamma-NGF and with a chimera in which the amino terminal region from EGF-BP was substituted. Two other chimeras containing either the middle or carboxyl terminal regions of gamma-NGF showed weaker ability to form 7S complexes. Thus, all chimeras containing two segments from gamma-NGF retained at least some ability to form the 7S complex. rEGF-BP reacted weakly with EGF, but the chimera composed of the amino and middle segments of EGF-BP and the carboxyl terminal segment of gamma-NGF formed a nativelike HMW-EGF complex. None of the other chimeras appeared to bind EGF. These results identify amino acid positions within each kallikrein that participate in strong growth factor interactions and demonstrate that, outside of active site contacts, different regions of the kallikreins are involved in the binding of EGF and beta-NGF, respectively.     

Biological and biochemical properties of nerve growth factor synthesized by mouse S-180 cells in culture

Nerve Growth Factor (NGF), a protein involved in the maintenance and differentiation of sensory and sympathetic neuronal cells [1], is synthesized by several different types of cells in culture [2-7]. In this paper, the biochemical and biological properties of NGF synthesized by a mouse S-180 sarcoma cell line were examined. These cells do not appear to produce the 7S-NGF molecule, a form of NGF found in high concentrations in the mouse submandibular gland [8]. The 7S-NGF is comprised of three distinct protein subunits named beta-NGF, alpha and gamma [9]. Although the S-180 cells do not produce 7S-NGF, the cells do synthesize one of the component subunits of 7S-NGF, the beta-NGF subunit. Biological, electrophoretic, immunological and molecular weight criteria were used to establish that the beta-NGF synthesized by the S-180 cells is very similar to the submandibular gland beta-NGF. The S-180 beta-NGF was bound to an unidentified binding component(s) which is not immunologically similar to either the alpha- or gamma-subunit. The functional significance of this interaction is not known.     

The expression of two kallikrein gene family members in the rat kidney

The mRNAs for two kallikrein gene family members expressed in the rat kidney have been characterized. One mRNA (PS) has previously been found in the pancreas and submaxillary gland and encodes true kallikrein. The second mRNA (K1) encodes a novel kallikrein-like enzyme expressed in the kidney and submaxillary gland that retains many of the key amino acid residues for the characteristic enzymatic cleavage specificity of kallikrein. Two oligonucleotide hybridization probes specific for the K1 mRNA demonstrate that the K1 mRNA is expressed in the kidney and submaxillary gland, but in none of the other eight tissues known to express one or more members of the rat kallikrein gene family. The K1 mRNA is the dominant kallikrein-related mRNA of the kidney, expressed at roughly 10 times the level of the true kallikrein (PS) mRNA. In the submaxillary gland the K1 mRNA is expressed at roughly one-fourth the level of true kallikrein mRNA.     

Differential effect of DHEA on mitogen-induced proliferation of T and B lymphocytes

Dehydroepiandrosterone (DHEA) is the predominant steroid hormone secreted by adrenal gland, and it has been proposed in recent years that DHEA has significant effects on immune function. We investigated the effect of DHEA (1 x 10(-5) - 1 x 10(-8)M) on proliferation of human T cells and B cells and on immunoglobulin production, a representative function of B cells. High doses of DHEA (1 x 10(-5)) significantly inhibited proliferation of peripheral blood mononuclear cells (PBMCs) and T cells induced by T cell mitogens hemagglutinin (PHA) and concanavalin A (Con A). Proliferation of PBMCs induced by B cell mitogens pokeweed mitogen (PWM) was increased by 1 x 10(-7) - 1 x 10(-6)M DHEA. Proliferation of PBMCs and B cells induced by Staphylococcus aureus Cowan strain I (SAC) was not significantly changed at any concentrations of DHEA. However, a concentration of 1 x 10(-7)M DHEA tended to potentiate their proliferation. This study suggested that DHEA acted on T and B lymphocytes differentially in immune system.     

Substance P enhances IL-2 expression in activated human T cells.

Jurkat and HUT 78 T cell lines, as well as peripheral blood human T cells activated with PHA plus PMA were used to investigate the capacity of substance P (SP) neuropeptide to regulate IL-2 production. By using Northern blot analysis and dosage of the IL-2 release in cell supernatants, we show that SP can act as cosignal with PHA + PMA to enhance the expression of specific IL-2 mRNA and IL-2 secretion in T cells. By using the N-terminal SP(1-4) or the C-terminal SP(4-11) fragments of the entire molecule, we show that the cosignal activity is carried by the C-terminal portion of SP. The SP and SP(4-11) optimal effects were observed at 10(-12) M and 10(-10) M when a broad range of concentrations from 10(-6) M to 10(-13) M was tested. The increase of IL-2 mRNA obtained with 10(-12) M of SP in the activated Jurkat cells was reduced by adding 10(-10) or 10(-9) M of the SP antagonist (D-Pro2,-D-Phe7,-D-Trp9)SP to the culture, indicating the specificity of SP action. The up-regulation observed when 10(-12) M of SP was applied together with the mitogens on Jurkat cells, persisted after a 16-h culture period, time at which the IL-2 mRNA signal is normally back to a minimum level when the mitogens are used alone. Furthermore, an induction of IL-2 mRNA accumulation, in a 2-h pulse, was obtained with 10(-12) M of SP on Jurkat cells previously activated with mitogens for 16 h.     

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.     

Evidence for the Presence of Nontranslated T7 Late mRNA in Infected F′(PIF+) Episome-Containing Cells

The inability of T7 to develop in cells of Escherichia coli containing F(+) or substituted F' episomes is a result of the failure to synthesize late proteins; no in vivo translation of mRNA species synthesized by the T7 RNA polymerase occurs. Further experiments have been performed to measure the amount of late mRNA in T7-infected F'(PIF(+)) cells. (We have designated the property of phage inhibition of F factors as PIF; the wild-type episome is therefore F'[PIF(+)].) T7 late proteins were synthesized in vitro by using a system programed with RNA extracted from T7-infected F(-) and F'(PIF(+)) cells. The T7 lysozyme, product of gene 3.5, and the gene 10 head protein were assayed. The following results were obtained: (i) mRNA capable of supporting in vitro synthesis of lysozyme and the gene 10 head protein is present in T7-infected F'(PIF(+)) cells; (ii) lysozyme mRNA extracted from T7-infected F'(PIF(+)) cells is present at 70 to 75% of the level found in T7-infected F(-) cells; (iii) gene 10 mRNA is present at 35 to 78% of the level found in T7-infected F(-) cells. No in vivo synthesis of either lysozyme or gene 10 protein can be detected in T7-infected F'(PIF(+)) cells although normal synthesis of these proteins occurs in F(-) cells. These findings confirm that the block in T7 development in F'(PIF(+)) cells results from the failure to translate late classes of T7 RNA.     

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.     

Rapid Isolation of the 7S-Nerve Growth Factor Complex and Its Subunits from Murine Submaxillary Glands and Saliva

7S-Nerve growth factor (NGF) and its alpha, beta-NGF, and gamma subunits have been purified from murine submaxillary glands and saliva by a combination of gel filtration on rigid polyvinyl gels, reversed-phase liquid chromatography on short alkyl chain supports (C4 columns), and ion-exchange chromatography on silica-based carboxymethyl columns. This technique is superior to previously used methods in that it is much more rapid and allows the purification of larger quantities of polypeptide from the same amount of starting material. Beta-NGF prepared with this method elicits the outgrowth of fibers of cells of a pheochromocytoma cell line (PC 12) in vitro, indicating that the biological activity is not impaired by the organic solvents and strong acids utilized for its isolation.     

大鼠颌下腺及其离体培养细胞脱氢表雄酮（DHEA）的免疫组织化学定位

用免疫组织化学ABC法,研究了颌下腺及无血清培养的颌下腺上皮细胞DHEA的定位,结果显示,大鼠颌下腺的浆液性腺泡的上皮细胞及各级导管上皮细胞均呈DHEA免疫反应阳性,无血清培养腺上皮细胞也呈DHEA免疫反应阳性,阳性物质分布于胞质,胞核呈阴性反应,此结果提示：大鼠颌下腺能自身合成DHEA,DHEA对消化功能可能具有重要的调节作用。     

Induction of osteoblast differentiation indices by statins in MC3T3-E1 cells

Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Simvastatin at 10(-7) M markedly increased mRNA expression for bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (OCN) in nontransformed osteoblastic cells (MC3T3-E1), while suppressing gene expression for collagenase-1, and collagenase-3. Extracellular accumulation of proteins such as VEGF, OCN, collagenase-digestive proteins, and noncollagenous proteins was increased in the cells treated with 10(-7) M simvastatin, or 10(-8) M cerivastatin. In the culture of MC3T3-E1 cells, statins stimulated mineralization; pretreating MC3T3-E1 cells with mevalonate, or geranylgeranyl pyrophosphate (a mevalonate metabolite) abolished statin-induced mineralization. Statins stimulate osteoblast differentiation in vitro, and may hold promise drugs for the treatment of osteoporosis in the future.     

Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells

The cholesterol-lowering drug, simvastatin, is a pro-drug of a potent 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and inhibits cholesterol synthesis in humans and animals. In addition, the bone effects of statins including simvastatin are being studied. We assessed the effects of simvastatin on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. Simvastatin enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the statin was observed at relatively low doses (significant at 10(-8) M and maximal at 10(-7) M). Northern blot analysis showed that the statin (10(-7) M) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. Simvastatin (10(-7) M) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 14 and 22 of culture. These results indicate that simvastatin has anabolic effects on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases such as osteoporosis.     

Demonstration and partial characterization of cytosol receptors for testosterone.

Androgen uptake was investigated in several peripheral organs after administration of (1,2,6,7 minus -3H)testosterone to castrated male rats. The animals were killed after 30 min, the organs were taken out, and the radioactivity was determined after tissue combustion. A relatively high accumulation of androgen was found in pancreas, adrenals, spleen, thigh muscle, kidneys, and liver in addition to the classical androgen target organs coagulation glands, seminal vesicles, prostate, preputial glands, and harderian glands. In a second serier of experiments, nuclear and cytosol fractions were prepared from prostate, seminal vesicles, coagulation glands, preputial glands, spleen, submaxillary glands, kidneys, and pancreas from castrated male rats give (1,2,6,7 minus -3H)testosterone, and these fractions were then characterized by thin-layer and radio-gas chromatography with respect to their patterns of labeled steroids. Only prostate and seminal vesicles were found to contain significant amounts of nuclear 5alpha-(-3H)dihydrotestosterone. The major nuclear androgen was (-3H)testosterone that was the only detectable labeled steroid in coagulation glands, preputial glands, and spleen and that constituted 70% or more of the nuclear radioactivity in seminal vesicles, submaxillary glands, kidneys, and pancreas. These results indicate that testosterone itself may be the predominant active androgen principle in vivo in most androgen target organs and that conversion to 5alpha-dihydrotestosterone is generally not a prerequisite for androgen activity. Using an ultrasensitive micromodification of isoelectric focusing (cf. M. Katsumata and A. S. Goldman (1974), Biochem. Biophys. Acta 359, 112. It was possible to show that cytosol from kidney; submaxillary gland, thigh muscle, and levator ani muscle and nuclei from kidney and submaxillary gland contained androgen-binding proteins with pI's in the region 4.6-5.1 ("4.6 minus 5.1 Complex"). This complex also formed in vitro after incubation of (1,2,6,7 minus -3H)testosterone with cytosol from kidney and submaxillary gland. (1,2,6,7 minus -3H)Testosterone was bound with high affinity to receptor proteins in cytosol from both kidney, submaxillary gland, and thigh muscle with dissociation constants of 5.0 x 10 minus -12 M (kidney), 3.3 x 10 mi;nus -11 M and 4.1 x 10 minus -10 M (two types of binding sites, submaxillary gland), 2.4 x 10 minus -12 M (thigh muscle) and 1.9 x 10 minus -12 M (levator ani muscle). The number of binding sites was in all cases between 1 and 20 fmol/mg of protein. On the basis of these results the hypothesis is presented that a common class of testosterone receptors is present in most organs and that these receptors can be detected both in vivo and in vitro provided methods sensitive enough are utilized.