RESUMO
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.
Assuntos
Fatores de Crescimento Neural/metabolismo , Neuroglia/metabolismo , Bioensaio , Linhagem Celular , Colífagos , Gânglios Espinais/efeitos dos fármacos , Fatores de Crescimento Neural/farmacologia , RadioimunoensaioRESUMO
Normal primary chick embryo fibroblast cultures product a nerve growth-promoting factor which cross-reacts with monospecfic antibody to pure male mouse submaxillary gland nerve growth factor (NGF). When taken together with the earlier demonstration that mouse L2 CELLS AND 3T3 cells also produce an NGF-like protein, these findings suggest that secretion of this factor may be a general property of fibroblast.
Assuntos
Fibroblastos/metabolismo , Fatores de Crescimento Neural/biossíntese , Animais , Células Cultivadas , Embrião de Galinha , Imunoensaio , CamundongosRESUMO
IgA is transported into external secretions by the polymeric Ig receptor (pIgR). Interferon-gamma (IFN-gamma), a major regulator of pIgR expression, has been shown to increase pIgR mRNA levels in HT-29 human colon carcinoma cells. To determine the molecular mechanisms of pIgR regulation, genomic DNA containing the 5'-flanking region of the human pIgR gene was isolated and a single start site of transcription in human intestinal epithelial cells was identified. Using chimeric reporter plasmids containing flanking regions of the pIgR gene, a segment of the pIgR promoter which is necessary and sufficient for induction of transcription by IFN-gamma in HT-29 cells was identified. Significantly, the pIgR promoter contains three motifs homologous to the interferon-stimulated response element (ISRE), two in the 5'-flanking region and one in exon 1 of the pIgR gene. The upstream ISREs bind nuclear protein(s) which are constitutively expressed by HT-29 cells, while the exon 1 ISRE binds interferon regulatory factor-1 (IRF-1), following stimulation with IFN-gamma. Furthermore, induction of the IRF-1 promoter by IFN-gamma correlates with induction of the pIgR promoter by IFN-gamma. It has previously been demonstrated that induction of pIgR mRNA by IFN-gamma, requires de novo protein synthesis. It is now shown that IRF-1 is not detected in nuclear extracts from HT-29 cells stimulated with IFN-gamma in the presence of cycloheximide, suggesting that de novo synthesis of IRF-1 is required for induction of pIgR transcription by IFN-gamma.
Assuntos
Regulação da Expressão Gênica/imunologia , Interferon gama/farmacologia , Receptores de Imunoglobulina Polimérica/genética , Transcrição Gênica/imunologia , Sequência de Bases , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Éxons/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HT29 , Humanos , Fator Regulador 1 de Interferon , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/imunologia , Receptores de Imunoglobulina Polimérica/biossíntese , Receptores de Imunoglobulina Polimérica/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
A modified assay for choline is described which is shorter, yields more consistent responses, and requires considerably less time, space, and equipment than the existing assays.
Assuntos
Bioensaio/métodos , Colina/análise , Estudos de Avaliação como Assunto , Neurospora crassa/crescimento & desenvolvimento , Neurospora crassa/metabolismoRESUMO
Studies from several laboratories have shown that mouse submandibular glands and mouse saliva contain nerve growth factor (NGF) as part of a high molecular weight oligomeric macromolecule composed of three different subunits, termed alpha, beta, and gamma. The beta-subunit is the nerve growth-promoting protein. The gamma-subunit is a serine protease class enzyme of highly restricted substrate specificity. The alpha-subunit has no known function. This high molecular weight form of nerve growth factor is also a Zn(II)-containing metalloprotein. In the present study, measurements of multiple physicochemical parameters have been used to deduce the subunit structure of high molecular weight NGF. Results demonstrate that it contains two alpha-, one beta- and one gamma-subunit together with one tightly bound Zn(II) ion per molecule.
Assuntos
Fatores de Crescimento Neural , Animais , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Camundongos , Estrutura Molecular , Peso Molecular , Fatores de Crescimento Neural/isolamento & purificação , Conformação Proteica , Serina , Glândula Submandibular/análise , Zinco/análiseRESUMO
The single, highly stable form of mouse submandibular gland nerve growth factor (NGF), prepared as described by Young et al. [(1978) Biochemistry 17, 1490--1498] is a protease of restricted specificity that can convert plasminogen to plasmin. In the absence of plasminogen, NGF is not fibrinolytic, nor does it hydrolyze casein at a measurable rate. Treatment of NGF with diisopropyl fluorophosphate inhibits its ability to activate plasminogen as well as its capacity to hydrolyze certain synthetic arginine esters. These results indicate that NGF is a member of the class of serine proteases. Since NGF is known to be secreted at high concentrations in mouse saliva, it may serve to activate plasminogen (with subsequent fibrinolysis) somewhere in the alimentary tract. Plasminogen activation is the only known action of NGF upon a biologically important non-neural substrate.
Assuntos
Fatores de Crescimento Neural/metabolismo , Peptídeo Hidrolases/metabolismo , Plasminogênio/metabolismo , Animais , Ativação Enzimática , Fibrinolisina , Humanos , Cinética , Masculino , Camundongos , Glândula Submandibular , Especificidade por Substrato , TripsinaRESUMO
The molecular size and stability of the nerve growth factor (NGF) secreted in culture by L cells have been studied by sedimentation and gel filtration chromatography. Results indicate that L cell NGF has a molecular weight of 160,000. It contains as part of its structure a protein component that is biologically, immunologically, and electrophoretically indistinguishable from the biologically active factor purified from mouse submandibular glands. However, unlike pure gland NGF, L cell NGF is highly stable in solution, and this finding indicates that L cell NGF is a form of the factor different from that previously described.
Assuntos
Células L/metabolismo , Fatores de Crescimento Neural , Animais , Bioensaio , Embrião de Galinha , Eletroforese em Gel de Poliacrilamida , Fatores de Crescimento Neural/isolamento & purificação , Fatores de Crescimento Neural/metabolismoRESUMO
The concept that the salivary gland of the mouse is an endocrine organ for nerve growth factor (NGF) has been reexamined. Serum concentrations of the protein have been measured by radioimmunoassay in male and female mice and in mice from which the submandibular glands were removed. In spite of the fact that the submandibular glands of male mice contained more NGF than did those of female mice, no sex differences in circulating concentrations of the factor were detected. Furthermore, serum concentrations of NGF did not change after submandibular gland removal or after administration of several autonomic agonists. These results indicate that the submandibular glands are not endocrine organs with respect to NGF. On the other hand, extremely high concentrations of the factor are normally secreted in mouse saliva at levels that reflect the sex differences in the amount of NGF present in the glands. This finding suggests that the salivary gland is an exocrine organ for NGF and that the protein may play a biological role in saliva.
Assuntos
Fatores de Crescimento Neural/metabolismo , Saliva/metabolismo , Glândula Submandibular/fisiologia , Animais , Bioensaio , Feminino , Isoproterenol/farmacologia , Masculino , Camundongos , Fatores de Crescimento Neural/sangue , Fenilefrina/farmacologia , Pilocarpina/farmacologia , Radioimunoensaio , Fatores Sexuais , Glândula Submandibular/efeitos dos fármacosRESUMO
Some molecular properties of the nerve growth factor (NGF) secreted in mouse saliva and that present in submandibular glands have been measured for comparison with previously studied forms of NGF. The results show that mouse saliva contains two biologically active NGF species. One has a molecular weight near 114,000, and the other, a molecular weight of 13,000. The larger form is being continuously degraded to yield the smaller one, probably as a result of a slow enzymatic process. Virtually identical results were obtained with crude submandibular gland extracts. The larger NGF is neither the well-known 7S NGF nor 2.5S NGF. Our results indicate that the larger salivary NGF is the naturally occurring form of NGF as it exists in the submandibular gland and as it is secreted in saliva. Its biological properties and its function in saliva, if any, remain to be elucidated.
Assuntos
Fatores de Crescimento Neural , Saliva/metabolismo , Glândula Submandibular/metabolismo , Animais , Camundongos , Peso Molecular , Fatores de Crescimento Neural/metabolismoRESUMO
Sedimentation and gel-filtration studies of mouse submandibular gland 7S-nerve growth factor (NGF) reveal that this complex dissociates to yield its components at concentrations much higher than those required to exhibit biological activity. Results further indicate that the alpha and gamma protein c omponents of the 7S-NGF complex probably play no role in its biological activity when tested in vitro. The dissociation behavior of 7S-NGF is quite different from the properties of very dilute solutions of the NGF secreted by mouse L cells and of that present in fresh, unpurified submandibular gland homogenates, since both of these proteins display high molecular weights at concentrations where 7s-NGF is fully dissociated. Thus, it could be that 7S-NGF is not the form in which NGF exists in the mouse submandibular gland.
Assuntos
Fatores de Crescimento Neural , Animais , Sítios de Ligação , Cromatografia em Gel , Células L , Substâncias Macromoleculares , Matemática , Camundongos , Peso Molecular , Radioimunoensaio , Glândula SubmandibularRESUMO
Transcytosis of polymeric Ig (pIg) by mucosal epithelial cells is mediated by the polymeric Ig receptor (pIgR). Here we describe the characterization of a 3095-bp mouse pIgR cDNA, which encodes a protein of 771 amino acids. Northern blot analysis detected a single mouse pIgR transcript of 3.9 kb, expressed at high levels in small intestine and liver, and at low levels in lung. Alignment of the amino acid sequences of mouse, rat, human, bovine, and rabbit pIgR revealed that functional regions of the molecule are conserved across species. In the extracellular region, conserved motifs include: a 23-amino acid pIg-binding site, 11 intradomain disulfide bonds, consensus sites for N-glycosylation, and a putative cleavage site at which the extracellular region of pIgR (secretory component) is released from the plasma membrane. A 10-amino acid sequence within the transmembrane region is highly conserved, possibly reflecting a mechanism for transmitting signals from the extracellular region to the cytoplasmic tail. Conservation within the cytoplasmic tail of pIgR is clustered in motifs that mediate polarized sorting, endocytosis, and transcytosis.
Assuntos
Camundongos/genética , Componente Secretório/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Bovinos/genética , Clonagem Molecular , DNA Complementar/genética , Genes , Glicosilação , Humanos , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , Processamento de Proteína Pós-Traducional , Coelhos/genética , Ratos/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Rat skeletal muscle cells and a cloned myogenic cell line synthesize and secrete in culture a molecule that is immunologically and biologically indistinguishable from the active form of nerve growth factor (NGF) from mouse submandibular gland. This protein can be detected in medium conditioned by muscle cells both before and after fusion and in the soluble fraction of muscle cell homogenates. Chromatographic data also reveal that the molecular properties of muscle cell NGF differ from those of the growth factor purified from mouse submandibular glands. Muscle cell NGF has a molecular weight between 140,000 and 160,000, whereas purified mouse gland NGF has a molecular weight of 26,000. The biologic function of muscle cell NGF is not known, although it could be that it plays some role relating to the association of nerves and muscle in vivo.