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N2a Cells (n2a + cell)
Selected AbstractsCloning and functional characterization of a novel connexin expressed in somites of Xenopus laevisDEVELOPMENTAL DYNAMICS, Issue 3 2005Teun P. De Boer Abstract Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (,62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like structures at the cell interfaces. Similar ectopic expression in neural N2A cells resulted in functional electrical coupling, displaying mild, asymmetric voltage dependence. We thus cloned a novel connexin from Xenopus laevis, strongly expressed in developing somites, with no apparent orthologue in mammals. Developmental Dynamics 233:864,871, 2005. © 2005 Wiley-Liss, Inc. [source] Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide libraryFEBS JOURNAL, Issue 21 2007Jeannine Mohrlüder 4-Aminobutyrate type A (GABAA) receptor-associated protein (GABARAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABAA receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra-Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca2+ -dependent chaperone and a Ca2+ -buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra-endoplasmic reticulum functions. By phage display screening of a randomized peptide library, peptides that specifically bind GABARAP were identified. Their amino acid sequences allowed us to identify calreticulin as a potential GABARAP binding protein. GABARAP binding to calreticulin was confirmed by pull-down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant Kd = 64 nm and a mean lifetime of the complex of 20 min. Thus, the interaction between GABARAP and calreticulin is the strongest so far reported for each protein. [source] Protection from MPTP-induced neurotoxicity in differentiating mouse N2a neuroblastoma cellsJOURNAL OF NEUROCHEMISTRY, Issue 3 2001Luigi A. De Girolamo We have shown previously that subcytotoxic concentrations of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) inhibit axon outgrowth and are associated with increased neurofilament heavy chain (NF-H) phosphorylation in differentiating mouse N2a neuroblastoma cells while higher doses (>,100 µm) cause cell death. In this work we assessed the ability of potential neuroprotective agents to alleviate both MPTP-induced cell death (cytotoxicity) and MPTP-induced NF-H phosphorylation/reduction in axon outgrowth (neurotoxicity) in N2a cells induced to differentiate by dbcAMP. The neurotoxic effects of MPTP occurred in the absence of significant alterations in energy status or mitochondrial membrane potential. The hormone oestradiol (100 µm) reduced the cytotoxic effect of MPTP, but blocked di-butyryl cyclic AMP (dbcAMP)-induced differentiation, i.e. axon outgrowth. Both the cytotoxic and neurotoxic effects of MPTP were reduced by the monoamine osidase (MAO) inhibitors deprenyl and, to a lesser extent, clorgyline. Alleviation of both neurotoxicity and cytotoxicity was also achieved by conditioned medium derived from rat C6 glioma cells. In contrast, whilst the p38 MAP kinase inhibitor, SB202190, protected cells against MPTP-induced neurotoxicity, it could not maintain cell viability at high MPTP exposures. In each case neuroprotection involved maintenance of the differentiating phenotype linked with attenuation of NF-H hyper-phosphorylation; the latter may represent a mechanism by which neuronal cells can moderate MPTP-induced neurotoxicity. The use of a simplified neuronal cell model, which expresses subtle biochemical changes following neurotoxic insult, could therefore provide a valuable tool for the identification of potential neuroprotective agents. [source] NADPH oxidase inhibitor diphenyliodonium abolishes lipopolysaccharide-induced down-regulation of transferrin receptor expression in N2a and BV-2 cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2006Katarina Reis Abstract The activation of cellular inflammatory response is tightly linked to induced production of reactive oxygen species (ROS) and nitric oxide (NO), which in turn have been identified as important regulators of cellular iron metabolism. In the present study, we have used the microglia cell line BV-2 and the neuroblastoma cell line N2a to study the regulatory effects of the microbial agent lipopolysaccharide (LPS) on the expression of the transferrin receptor (TfR) and ferritin in cell lines with different characteristics. The receptor mainly responsible for LPS recognition is the Toll-like receptor 4 (TLR4) that triggers a variety of intracellular signalling cascades leading to the induction of transcription of target genes involved in the innate immune response. Among the pathways to be activated is the MAPK cascade leading to the activation of nuclear factor-,B that induces transcription of a variety of genes, e.g., inducible nitric oxide synthase (iNOS). The TLR4-mediated LPS response also induces the production of ROS through a mechanism(s) suggested to involve the activation of NADPH oxidase(s). This study shows that exposure of BV-2 and N2a cells to LPS results in decreased TfR protein levels and increased H-ferritin mRNA levels. The LPS down-regulatory effect on TfR protein expression is abolished by the NADPH oxidase inhibitor diphenyliodonium (DPI) but is not affected by the free radical scavenger N-acetyl-L-cysteine (NAC) or the iNOS inhibitor aminoguanidine (AG). The increased H-ferritin mRNA levels in response to LPS are not affected by DPI, NAC, or AG. © 2006 Wiley-Liss, Inc. [source] Loss of lipopolysaccharide-induced nitric oxide production and inducible nitric oxide synthase expression in scrapie-infected N2a cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2003Heléne Lindegren Abstract In scrapie-infected cells, the conversion of the cellular prion protein to the pathogenic prion has been shown to occur in lipid rafts, which are suggested to function as signal transduction platforms. Neuronal cells may respond to bacterial lipopolysaccharide (LPS) treatment with a sustained and elevated nitric oxide (NO) release. Because prions and the major LPS receptor CD14 are colocalized in lipid rafts, the LPS-induced NO production in scrapie-infected neuroblastoma cells was studied. This study shows that LPS induces a dose- and time-dependent increase in NO release in the murine neuroblastoma cell line N2a, with a 50-fold increase in NO production at 1 ,g/ml LPS after 96 hr, as measured by nitrite in the medium. This massive NO release was not caused by activation of the neuronal NO synthase (nNOS), but by increased expression of the inducible NOS (iNOS) mRNA and protein. However, in scrapie-infected N2a cells (ScN2a), the LPS-induced NO production was completely abolished. The absence of LPS-induced NO production in ScN2a was due not to abolished enzymatic activity of iNOS but to a complete inhibition of the LPS-induced iNOS gene expression as measured by Western blot and RT-PCR. These results indicate that scrapie infection inhibits the LPS-mediated signal transduction upstream of the transcriptional step in the signaling cascade and may reflect the important molecular and cellular changes induced by scrapie infection. © 2002 Wiley-Liss, Inc. [source] | ||||||||||