Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0027819 (neuroblastoma)
 27,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rabies is an endemic, fatal zoonotic disease in the developing countries. Prevention and post-exposure therapy require safe and efficacious vaccines. The vaccine potency depends on the amount of immunogenic rabies viral glycoprotein antigen in the vaccine preparation. In order to estimate the rabies viral glycoprotein antigen, a specific monoclonal antibody was developed and used in an immuno-capture ELISA (IC-ELISA). The monoclonal antibody binds a conformational epitope on the natively folded rabies viral glycoprotein as indicated by specific, membrane fluorescence on unfixed, rabies virus infected murine neuroblastoma (MNA) cells and glycoprotein gene encoding plasmid transfected COS cells. In addition, the monoclonal antibody competes with and blocks a glycoprotein antigen site III binding monoclonal antibody (mAb-D1, Institut Pasteur, Paris, France). The monoclonal antibody was used in an IC-ELISA using an in-house standard to quantify the rabies viral glycoprotein antigen in 12 vaccine preparations with potency values ranging from 4 to 18 IU. The results indicated a good correlation with the NIH mouse potency assay (r=0.83). The immuno-capture ELISA described in this study can be used to quantify the immunogenic rabies viral glycoprotein antigen in the inactivated rabies viral antigen preparation in a simple and rapid format, which enables better vaccine formulation.
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PMID:A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture. 1618 54

Expression of VPAC and PAC1 receptor isoforms was determined in six neuroblastoma cell lines as well as in human embryonic and adult brain using reverse transcriptase PCR and quantitative PCR. PAC1 receptor splice variants missing a 21 amino acid sequence in the amino terminal domain were found to be the major receptor variants in the neuroblastoma cell lines and also were highly expressed in embryonic brain compared to adult brain. In four of the neuroblastoma cell lines, VIP and PACAP stimulated cyclic AMP production with different potencies and levels of maximal stimulation. High potency and greatest maximal stimulation of cyclic AMP for each peptide were recorded in SH-SY5Y cells, indicating the presence of high affinity VIP and PACAP receptors. Further characterization of specific VPAC and PAC1 receptor isoforms was carried out in the SH-SY5Y cell line, where along with known PAC1 receptor splice variants and the VPAC2 receptor, a number of novel PAC1 receptor splice variants were identified. The comparatively low level expression of the VPAC2 receptor along with the poor responsiveness of SH-SY5Y cells to the VPAC2 receptor-specific agonist Ro 25-1553 indicated that this receptor did not contribute significantly to the observed VIP responses. When the individual PAC1 receptor isoforms were expressed in COS 7 cells, the ability of VIP to activate cyclic AMP production was increased more than 50-fold at the majority of the PAC1 receptor variants lacking the 21 amino acid amino terminal domain sequence compared to those with the complete domain. Smaller changes were seen in the potency of PACAP-38. Similar trends were seen with inositol phosphate responses, where in each case agonist potencies were lower than for cyclic AMP production. The results of this study show that the combination of different amino terminal and intracellular loop 3 splicing variants in the PAC1 receptor dictates the ability of agonists, particularly VIP, to activate signaling pathways. VIP has considerably greater potency at most PAC1 receptors with the short amino terminal domain, and these therefore may mediate physiological effects of both VIP and PACAP. Furthermore, there may be a phenotypic switch in the expression of different PAC1 receptor amino terminal splice variants between embryonic and mature nervous system, indicating that regulation of this event may have an important role in VIP/PACAP function, particularly in the developing nervous system.
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PMID:Characterization of novel splice variants of the PAC1 receptor in human neuroblastoma cells: consequences for signaling by VIP and PACAP. 1622 89

Transactivation of epidermal growth factor receptor (EGFR) by G protein-coupled receptors (GPCRs) has been attributed to the activation of matrix metalloproteases (MMPs) and the release of EGF family ligands such as HB-EGF. This mode of transactivation leads to signalling downstream of EGFR which is indistinguishable from that induced by the ligand. Here we provide evidence that in the COS-7 cell model EGFR transactivation via the muscarinic M2 receptor (M2R) is independent of MMPs and results in an incomplete EGFR signalling including ERK and Akt but not PLCgamma1. Using dominant-negative mutants of c-Src and Fyn and Src-deficient SYF cells as well as by co-immunoprecipitation studies, we can demonstrate that the M2R-mediated transactivation of EGFR specifically involves Fyn but not c-Src or Yes. This specific role of Fyn can be verified in SH-SY5Y human neuroblastoma cells with endogenously expressed M2 receptors.
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PMID:Muscarinic M2 receptors mediate transactivation of EGF receptor through Fyn kinase and without matrix metalloproteases. 1633 76

Cocaine, amphetamines and other psychostimulants inhibit synaptic dopamine uptake by interfering with dopamine transporter (DAT) function. The resultant potentiation of dopaminergic neurotransmission is associated with psychostimulant addiction. Fluctuations in dopamine uptake inhibition potency (DUIP) were observed for classical DAT blockers including cocaine, mazindol, methylphenidate (Ritalintrade mark) and benztropine in CHO cells expressing wild type DAT; cocaine potency also decreased in DAT-expressing non-neuronal COS-7 cells and neuronal N2A neuroblastoma cells. In contrast, the DAT substrate (+)-amphetamine did not display this DUIP fluctuation. In parallel experiments, no fluctuation was observed for the apparent binding affinities of these 5 drugs. The DUIP decrease appeared to correlate with an increase in cell surface DAT expression level, as measured by B(max) values and confocal microscopy. The fact that the DUIP profile of amphetamine diverged from that of the classical DAT blockers is consistent with the idea of fundamental differences between the mechanisms of abused psychostimulant DAT substrates and inhibitors. Identification of the cellular factors that underlie the DAT inhibitor DUIP fluctuation phenomenon may be relevant to anti-psychostimulant drug discovery efforts.
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PMID:Fluctuation of the dopamine uptake inhibition potency of cocaine, but not amphetamine, at mammalian cells expressing the dopamine transporter. 1716 38

The gene of mammalian acetylcholinesterase (AChE) generates multiple molecular forms, by alternative splicing of its transcripts and association of the tailed variant (AChET) with structural proteins. In the mammalian brain, the major AChE species consists of AChET tetramers anchored to the cell membrane of neurons by the PRiMA protein (Perrier et al., 2002). Stress and anticholinesterase inhibitors have been reported to induce rapid and long-lasting expression of the readthrough variant (AChER) in the mouse brain (Kaufer et al., 1998). In the readthrough transcript, there is no splicing after the last exon encoding the catalytic domain, so that the entire alternatively spliced 3' region is maintained. It encodes a C-terminal peptide with no specific interaction properties: COS cells transfected with AChER produce a soluble, nonamphiphilic monomeric form. We quantified AChER and total AChE expression in the mouse brain after an immobilization stress and after heat shock in neuroblastoma cells, and compared the observed effects with those induced by irreversible AChE inhibition (Perrier et al., 2005).
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PMID:Readthrough acetylcholinesterase expression remains minor after stress or exposure to inhibitors. 1719 35

The amyloid precursor protein (APP) metabolism is central to pathogenesis of Alzheimer's disease (AD). Parenchymal amyloid deposits, a neuropathological hallmark of AD, are composed of amyloid-beta peptides (Abeta). Abeta derives from the amyloid precursor protein (APP) by sequential cleavages by beta- and gamma-secretases. Gamma-secretase cleavage releases the APP intracellular domain (AICD), suggested to mediate a nuclear signaling. Physiologically, AICD is seldom detected and thus supposed to be rapidly degraded. The mechanisms responsible of its degradation remain unknown. We used a pharmacological approach and showed that several alkalizing drugs induce the accumulation of AICD in neuroblastoma SY5Y cell lines stably expressing APP constructs. Moreover, alkalizing drugs induce AICD accumulation in naive SY5Y, HEK and COS cells. This accumulation is not mediated by the proteasome or metallopeptidases and is not the result of an increased gamma-secretase activity since the gamma-secretase cleavage of Notch1 and N-Cadherin is not affected by alkalizing drug treatments. Altogether, our data demonstrate for the first time that alkalizing drugs induce the accumulation of AICD, a mechanism likely mediated by the endosome/lysosome pathway.
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PMID:Intracellular pH regulates amyloid precursor protein intracellular domain accumulation. 1720 30

Mutations in the PARKIN (PARK2) gene have been found in the majority of early-onset familial Parkinson's disease (PD) patients with autosomal recessive juvenile parkinsonism (ARJP). Parkin protein functions as an ubiquitin (E3) ligase that targets specific proteins for degradation in the 26S proteasome. Here, based on a mass spectrometry analysis of the human dopaminergic neuroblastoma-derived cell line SH-SY5Y that over-expresses parkin, we found that parkin may suppress cofilin phosphorylation. LIM Kinase 1 (LIMK1) is the upstream protein that phosphorylates cofilin, an actin depolymerizing protein. Thus, we postulated a possible connection between parkin and LIMK1. Our studies in other cell lines, using co-transfection assays, demonstrated that LIMK1 and parkin bind each other. LIMK1 also interacted with previously known parkin interactors Hsp70 and CHIP. Parkin enhanced LIMK1-ubiquitination in the human neuroblastoma-derived BE(2)-M17 cell line, but not in the human embryonic kidney-derived HEK293 cell line. In fact, parkin-over-expression reduced the level of LIMK1-induced phosphocofilin in the BE(2)-M17 cells but not in the HEK293 cells. Additionally, in simian kidney-derived COS-7 cells, parkin-over-expression reduced LIMK1-induced actin filament accumulation. LIMK1 in cultured cells regulates parkin reversibly: LIMK1 did not phosphorylate parkin but LIMK1 overexpression reduced parkin self-ubiquitination in vitro and in HEK293 cells. Furthermore, in the cells co-transfected with parkin and p38, LIMK1 significantly decreased p38-ubiquitination by parkin. These findings demonstrate a cell-type dependent functional interaction between parkin and LIMK1 and provide new evidence that links parkin and LIMK1 in the pathogenesis of familial PD.
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PMID:Parkin interacts with LIM Kinase 1 and reduces its cofilin-phosphorylation activity via ubiquitination. 1751 23

The secreted fragment of the amyloid precursor protein (sAPPalpha) generated following cleavage by alpha-secretase is an important mediator of cell function and is both neurotrophic and neuroprotective. HEK 293T cells have been stably integrated with a fragment of the APP gene to produce and secrete either sAPPalpha, or the alternative cleavage product sAPPbeta. Heparin binding domains on the proteins have been utilised to develop a one-step fast-performance-liquid-chromatography (FPLC) purification of sAPPs from the conditioned media. Immunoblotting analyses with a sAPP specific antibody coupled with highly sensitive silver staining techniques have validated the expression and purification strategy. Functional activity of the purified fragments was demonstrated by their ability to protect COS-7 and SH-SY5Y (neuroblastoma) cells against the adverse effects of glucose deprivation in a cell viability assay. The purified sAPPs also activated the NFkappaB transcription factor in COS-7 cells transfected with a luciferase reporter plasmid, with sAPPalpha the more potent activator as expected. The simple protocol to produce these mammalian expressed proteins will facilitate their use as potential neuropharmacological reagents in the elucidation of biochemical pathways modulated by sAPPs, and in the study of Alzheimer's disease mechanisms in general.
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PMID:Production, purification and functional validation of human secreted amyloid precursor proteins for use as neuropharmacological reagents. 1753 17

Retinoic acid (RA) treatment of SH-SY5Y neuroblastoma cells results in activation of phosphatidylinositol-3-kinase (PI3K) signaling pathway, and this activation is required for RA-induced differentiation. Here we show that RA activates PI3K and ERK1/2 MAPK signaling pathways through a rapid, nongenomic mechanism that does not require new gene transcription or newly synthesized proteins. Activation of PI3K by RA appears to involve the classical nuclear receptor, retinoic acid receptor (RAR), on the basis of the pharmacological profile of the activation, loss, and gain of function experiments with mouse embryo fibroblast-RAR(alpha beta gamma)(L-/L-) null cells, and the physical association between liganded RAR and PI3K activity. The association of RAR with the two subunits of PI3K was differentially regulated by the ligand. Immunoprecipitation experiments performed in SH-SY5Y cells showed stable association between RARalpha and p85, the regulatory subunit of PI3K, independently of the presence of RA. In contrast, ligand administration increased the association of p110, the catalytic subunit of PI3K, to this complex. The intracellular localization of RAR proved to be relevant for PI3K activation. A chimerical RAR fusing c-Src myristylation domain to the N terminus of RARalpha (Myr-RARalpha) was targeted to plasma membrane. Transfection of Myr-RARalpha to mouse embryo fibroblast-RAR(alpha beta gamma)(L-/L-) null cells and COS-7 cells results in strong activation of the PI3K signaling pathway, although both in the absence as well in the presence of RA. Our results support a mechanism in which ligand binding to RAR would play a major role in the assembly and intracellular location of a signaling complex involving RAR and the subunits of PI3K.
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PMID:Rapid, nongenomic actions of retinoic acid on phosphatidylinositol-3-kinase signaling pathway mediated by the retinoic acid receptor. 1759 18

The peptidyl prolyl cis-trans isomerase Pin1 and the Inhibitor of Apoptosis Protein (IAP) Survivin are two major proteins involved in cancer. They both modulate apoptosis, mitosis, centrosome duplication and neuronal development but until now no functional relationship has been reported between these two proteins. We tested Pin1-induced regulation of Survivin in neuroblastoma cells. Pin1 overexpression in SY5Y neuroblastoma cells decreased Survivin levels. Immunocytochemical studies indicated that they partially co-localized in interphase and mitotic cells. Co-immunoprecipitation further demonstrates the existence of a Pin1/Survivin complex. Pin1-induced effect on Survivin was confirmed in COS cells. RT-PCR and mutagenesis experiments suggested that this Pin1-induced decrease of Survivin occurred at the protein level. Survivin downregulation depended on the binding ability of Pin1 but was not related to the single Thr-Pro site, suggesting an indirect relationship into a protein complex. Finally, this functional regulation of Survivin by Pin1 is reciprocal since Pin1 silencing led to an increase in Survivin levels. The characterization of this functional relationship between Pin1 and Survivin might help to better understand mitosis control and cancer mechanisms.
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PMID:The peptidyl prolyl cis/trans isomerase Pin1 downregulates the Inhibitor of Apoptosis Protein Survivin. 1762 54


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