Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UMLS:C0027819 (neuroblastoma)
27,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Alzheimer amyloid precursor (APP) protein is a member of a family of glycoproteins that includes the amyloid precursor-like proteins (APLPs). Previously, we showed that in C6 glioma cell cultures, secreted APP nexin II occurs as the core protein of a chondroitin sulfate proteoglycan (CSPG). Here, we report that among seven untransfected cell lines, expression of secreted APP CSPG was restricted to two cell lines of neural origin, namely, C6 glioma and Neuro-2a neuroblastoma (N2a) cells. Addition of dibutyryl cyclic AMP in N2a cultures, a treatment that induces the neuronal phenotype in these cells, resulted in a significant reduction in the amount of the secreted APP CSPG, although secretion of APP was only marginally affected. Growth in the presence of serum increased the size of the secreted APP CSPG, suggesting that the number and/or length of the chondroitin sulfate (CS) chains attached to the core APP varies with growth conditions. Extensive mapping with epitope-specific antibodies suggested that a CS chain is attached within or proximal to the A beta sequence of APP. In contrast to the restricted expression of the APP CSPG, expression of secreted APLP2 CSPGs was observed in all cell lines examined. After chondroitinase treatment, two core proteins of approximately 100 and 110 kDa were obtained that reacted with an APLP2-specific antiserum, suggesting that non-transfected cell lines contain at least two endogenous APLP2 CSPGs, probably derived by alternative splicing of the APLP2 KPI domain. The fraction of the APLP2 proteins in the CSPG form was dependent on the particular cell line examined.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of the chondroitin sulfate proteoglycans of amyloid precursor (appican) and amyloid precursor-like protein 2. 761 33

Recombinant rat glia-derived nexin was expressed in insect cells using the baculovirus system. The kinetics for the inhibition of thrombin by this recombinant material were indistinguishable from those observed with natural glia-derived nexin and recombinant nexin expressed in yeast. In addition, the dependence of the rate of inactivation on the concentration of heparin was similar for the three preparations. At the optimal heparin concentration, the association rate constant was 330-fold higher than that observed in the absence of heparin. A putative heparin-binding site is found in glia-derived nexin between residues 71 and 86; heparin-binding sites are found in homologous regions of antithrombin III and heparin cofactor II. Lysines in this region were mutated to glutamates, and the kinetics for the inhibition of thrombin by mutant proteins were determined. Concurrent mutation of all seven lysines in this region (residues 71, 74, 75, 78, 83, 84, and 86) did not affect the rate constant for the association of glia-derived nexin with thrombin in the absence of heparin, but it resulted in complete loss of the heparin acceleration of the rate of association. Mutations of residues 83, 84, and 86 together also caused a marked decrease in the acceleration by heparin of the reaction between glia-derived nexin and thrombin. These results support the hypothesis that the heparin-binding sites of glia-derived nexin, antithrombin III, and heparin cofactor II are found in homologous regions of the molecules. Heparin was also found to potentiate the ability of wild-type glia-derived nexin to inhibit the thrombin-induced retraction of neurites from neuroblastoma NB2a cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Localization of the heparin-binding site of glia-derived nexin/protease nexin-1 by site-directed mutagenesis. 801 37

The characteristic features of Alzheimer's disease (AD) include a high density of beta-amyloid-containing plaques in the cerebral cortex and the loss of basal forebrain cholinergic neurons. Amyloid beta-protein (A beta, Mr. approximately 4.5 kDa) is derived from a family of large (Mr. approximately 110-140 kDa) beta-amyloid precursor proteins (APP) which are integral membrane glycoproteins consisting of a large extracytoplasmic domain, a transmembrane domain, and a short cytoplasmic tail. Secreted derivatives of APP lacking the cytoplasmic tail, transmembrane domain, and a small portion of the extracellular domain are generated by the proteolytic processing of full length APP by a family of proteolytic enzymes known as APP secretases. Using cell cultures, we investigated the possibility that APP processing can be regulated by a centrally active cholinesterase inhibitor, tacrine, which has recently been shown to improve memory and cognitive functions in patients with AD. We analyzed the level of APP in glial, fibroblast, pheochromocytoma (PC12), and neuroblastoma cells by immunoblotting cell lysates and conditioned media. Normal levels of secretion of soluble APP derivatives by cells into conditioned media were severely inhibited by treating cells with tacrine. A similar decrease after treatment with tacrine was observed when neuroblastoma and PC12 cells were pretreated with either growth factors, phorbol ester, or retinoic acid. To determine whether the effect of tacrine on APP levels was specific or a more general phenomenon affecting other proteins, we measured the level of heat shock protein-70 (HSP-70) and another secretory protein, protease nexin-1 (PN-1). Tacrine treatment did not alter the level of HSP-70 in cell extracts and tacrine affected mildly the secretion of PN-1. Thus, the processing of HSP and PN-1, unlike APP, was not severely affected by treating cells with tacrine. Our results suggest that tacrine may inhibit an acetylcholinesterase-associated proteolytic activity involved in the secretion of APP, which results in less secretion of soluble APP into the conditioned media from tacrine treated cells. These results demonstrate that tacrine regulates APP secretion in cell cultures and suggest the possibility that tacrine therapy of Alzheimer's disease may, in the longer term, have effects on the process of A beta deposition.
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PMID:Tacrine alters the secretion of the beta-amyloid precursor protein in cell lines. 804 78

Glia-derived nexin/protease nexin-1 (GDN/PN-1) is a serine protease inhibitor that is secreted by glial cells and fibroblasts in culture. In the adult mammalian nervous system it has been shown to be expressed in the olfactory system and by some glial cells in response to neuronal injury. In situ hybridization and immunocytochemical studies were performed to identify the structures expressing GDN/PN-1 in the developing and adult rat brain. In contrast to a transient widespread expression during pre- and postnatal development, some brain structures constitutively express GDN/PN-1. These include the olfactory nerve layer of the olfactory bulb, basal forebrain, striatum, pyramidal neurons of layer V in the cortex, thalamic nuclei, pars compacta of the substantia nigra, inferior and superior colliculi, and deep cerebellar nuclei. All of these parts, excluding the olfactory nerve layer, are characterized by a high neuronal cell density. Neurons in these regions were immunoreactive for GDN/PN-1. Furthermore GDN/PN-1 expression in cell lines showed that the active protein was synthesized and secreted from B104 but not from NB2a neuroblastoma cells. Although GDN/PN-1 has only been reported to be synthesized by glia, the results presented here demonstrate that in addition, a subset of neurons express this protease inhibitor.
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PMID:Glia-derived nexin/protease nexin-1 is expressed by a subset of neurons in the rat brain. 815 33

The C6 glial cell line has been used as a model cell system for the investigation of new glial produced neurotrophic and neurotropic molecules. By using the C6 cell line grown in a defined medium on collagen, this laboratory has isolated a distinct neurite promoting factor (NPF) that is potentiated by the presence of collagen (CPNPF). We have observed that C6 cells cultured in a defined medium on collagen (rat type-I) slowed their growth rate and expressed an astrocytic- or oligodendrocytic-like morphology. CPNPF, at this state of purity, appears to be a distinct NPF which induces neurite outgrowth (neurites of 1 or more somal diameters) in PC12 cells. These neurite promotion effects, however, appear to support the neuron morphology for only a short period (4 days) of time without the presence of neurotrophic factor (NTF). The neurite promoting activity is ineffective in inducing neurite outgrowth using mouse neuroblastoma cells (neuro-2a). CPNPF appears to be a heat stable protein whose activity does not depend on the presence of intact collagen, heparin sulfate proteoglycan (HSPG), or chondroitin sulfate proteoglycan (CSPG). Exposure to dissociative conditions results in a loss of neurite promoting activity. CPNPF is not a glycoprotein that contains an accessible alpha-D-mannopyranosyl, alpha-D-glucopyranosyl, or a sterically related residue (hydroxyl groups in the C-3,4, and 5 positions). Although these residues are not present on all glycoproteins, it does indicate that CPNPF is most likely not a glycoprotein. CPNPF activity is not blocked by neutralizing antibodies directed toward NGF, beta-FGF, IL-1 beta, IL-6, TGF-beta 2, TGF-beta 1.2, TGF-beta 3, TGF-beta 5, or EGF. CPNPF appears to either be oligomeric protein or a complex of proteins. On the basis of indirect evidence, it does not appear to be glial derived protease nexin-I. The alteration in morphology of the C6 glial cell line by serum-free conditions in the presence of collagen may have induced the production of a potentially new NPF not seen by previous investigators.
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PMID:Identification of a collagen potentiated neurite promoting factor isolated from C6 glioma cells. 836 Sep 47

The clotting protease thrombin might contribute to cell damage following brain injury by its ability to retract processes on neurons and astrocytes. Protease nexin-1 (PN-1), a potent inhibitor of thrombin, is localized around cerebral blood vessels where it may protect these cells from extravasated thrombin during injury or alteration of the blood-brain barrier. Here we examined the effects of several injury-related factors on the regulation of PN-1 in cultured brain cells. Interleukin-1, tumor necrosis factor-alpha, and transforming growth factor-beta stimulated the secretion of PN-1 by the neuroblastoma cell line SK-N-SH. This cell line comprises both neuronal and glial cells. Analyses using cloned derivatives of these two cell types showed that PN-1 was secreted by the glial cells; PN-1 secretion was stimulated 90-fold by interleukin-1, 15-fold by tumor necrosis factor-alpha, 10-fold by tumor growth factor-beta, and 4-fold by platelet-derived growth factor. Measurements of newly synthesised PN-1 demonstrated that these factors produced an equivalent stimulation of PN-1 synthesis. The neuronal cells secreted two thrombin-binding proteins distinct from PN-1. Interactions between these two cell types regulated the secretion of PN-1 and the two thrombin-binding proteins.
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PMID:Regulation of protease nexin-1 synthesis and secretion in cultured brain cells by injury-related factors. 842 47

In order to develop in vitro models of CNS injury, astrocytes have been mechanically injured in culture to study reactive astrocytosis. However, scratch injury models of pure neuronal cultures have not yet been exploited to study programmed cell death (PCD). For this study, we examined model motor neurons (NSC19 cells) in culture and found time-dependent cell death in proximity (within 2.5 mm) to a physical scratch injury. Injury-induced cell death was apoptotic verified by positively-stained nuclei using both the in situ end-labeling (ISEL) procedure and Hoechst 33342. Unexpectedly, cells proximal to the injury site were not affected by the injury until 3 days later suggesting that adjacent motor neuron loss was dependent on a 'death signal' produced by direct injury to sister neurons. 'Executioners' in apoptosis include free radicals, cell cycle kinases and cysteine proteases (caspases). Extracellular serine proteases, such as thrombin and granzyme B, may activate such intracellular pathways and several inhibitors (serpins), such as CrmA, are effective in blocking apoptosis. Since protease nexin I (PNI), a serpin homologous with CrmA, prevents apoptosis of lumbar motor neurons and is increased after nerve injury, we examined mRNA by RT-PCR for PNI expression. Of interest, although we were unable to find significant levels of PNI message in NSC19 cells, we did detect it in the parent neuroblastoma.
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PMID:Apoptotic, injury-induced cell death in cultured mouse murine motor neurons. 925 55

Increasing evidence indicates several roles for thrombin-like serine proteases and their cognate inhibitors (serpins) in normal development and/or pathology of the nervous system. In addition to its prominent role in thrombosis and/or hemostasis, thrombin inhibits neurite outgrowth in neuroblastoma and primary neuronal cells in vitro, prevents stellation of glial cells, and induces cell death in glial and neuronal cell cultures. Thrombin is known to act via a cell surface protease-activated receptor (PAR-1), and recent evidence suggests that rodent neurons express PAR-1. Previously, we have shown that the thrombin inhibitor, protease nexin-1, significantly prevents neuronal cell death both in vitro and in vivo. Here we have examined the effects of human alpha-thrombin and the presence and/or activation of PAR-1 on the survival and differentiation of highly enriched cultures of embryonic chick spinal motoneurons. We show that thrombin significantly decreased the mean neurite length, prevented neurite branching, and induced motoneuron death by an apoptosis-like mechanism in a dose-dependent manner. These effects were prevented by cotreatment with hirudin, a specific thrombin inhibitor. Treatment of the cultures with a synthetic thrombin receptor-activating peptide (SFLLRNP) mimicked the deleterious effects of thrombin on motoneurons. Furthermore, cotreatment of the cultures with inhibitors of caspase activities completely prevented the death of motoneurons induced by either thrombin or SFLLRNP. These findings indicate that (1) embryonic avian spinal motoneurons express functional PAR-1 and (2) activation of this receptor induces neuronal cell degeneration and death via stimulation of caspases. Together with previous reports, our results suggest that thrombin, its receptor(s), and endogenous thrombin inhibitors may be important regulators of neuronal cell fate during development, after injury, and in pathology of the nervous system.
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PMID:Thrombin perturbs neurite outgrowth and induces apoptotic cell death in enriched chick spinal motoneuron cultures through caspase activation. 971 58

The genes regulating the induction of differentiation in neurons are not definitively known. Some neuronal tumors retain the ability to differentiate into mature, functional neurons in response to pharmacological agents, despite the presence of genetic anomalies. We hypothesized that some of the genes whose expression is altered between undifferentiated and differentiated states may be those responsible for inducing differentiation. To investigate this, we used a mouse neuroblastoma (NB) cell line, NBP(2), in which > or =90% of the cells in the culture terminally differentiate upon elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels. Gene expression was analyzed using cDNA array blots containing 588 known genes. mRNA from cultures of undifferentiated and differentiated NB cells was used to make cDNA probes for blot hybridization. We identified several genes that are predominantly expressed in either undifferentiated or differentiated NB cells. In addition, numerous genes are moderately up- or down-regulated during differentiation of NB cells. We identified the N-myc protooncogene, cyclin B1, and protease nexin 1 as genes that are expressed in undifferentiated NB cells and whose levels are significantly down-regulated upon differentiation. In contrast, the c-fes and c-fos protooncogenes and the RAG-1 gene activator are genes whose expression is significantly up-regulated during differentiation of NB cells. These findings were confirmed by RT-PCR analysis. The transcript size and expression level of N-myc, cyclin B1, protease nexin 1, c-fes, and c-fos were verified by Northern blotting. These genes may represent key mediators involved in the regulation of NB cell differentiation.
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PMID:Identifying genes involved in regulating differentiation of neuroblastoma cells. 1131 75

Protease Nexin-1, a 43-kDa glycoprotein, is a major physiological thrombin inhibitor involved in the modulation of nerve cell plasticity. Recombinant rat Protease Nexin-1 (rPN-1) was efficiently produced in Escherichia coli using a T7 RNA polymerase based expression system and purified by heparin-sepharose affinity chromatography yielding 3 mg of protein per liter of cell culture. The purity and chemical identity of rPN-1 were assessed by SDS-PAGE, Reverse Phase- High Performance Liquid Chromatography, mass spectrometry and two-dimensional-gel electrophoresis. Conformational analysis by circular dichroism and fluorescence spectroscopy revealed the presence of mixed alpha/beta secondary structure and the prevailing localization of Trp-residues in rather polar environments. Fluorescence titration of rPN-1 with heparin indicated that rPN-1 binds heparin with high affinity. Furthermore, the formation of a SDS-stable 1:1 thrombin-rPN-1 complex, monitored by SDS-PAGE, confirmed the native-like structure of rPN-1. Finally, the cellular effects of rPN-1, such as its ability to promote neurite outgrowth in neuroblastoma cells, were found to be very similar to those elicited by natural PN-1. Altogether, our results demonstrate that glycosylation does not alter neither structure nor function of PN-1 and that E. coli is a suitable expression system for obtaining milligram quantities of pure and fully active rPN-1 for structural and functional studies.
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PMID:Conformational and biochemical characterization of a biologically active rat recombinant Protease Nexin-1 expressed in E. coli. 1916 25


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