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Mauricio Montal - One of the best experts on this subject based on the ideXlab platform.

  • voltage gated sodium channels expressed in the human cerebellar Medulloblastoma Cell Line te671
    Molecular Brain Research, 1990
    Co-Authors: Franco Gambale, Mauricio Montal
    Abstract:

    Abstract A characterization of the properties of voltage-gated sodium channels expressed in the human cerebellar Medulloblastoma Cell Line TE671 is presented. Membrane currents were recorded under voltage clamp conditions using the patch clamp technique in both the whole-Cell and the excised-patch configurations. Macroscopic sodium currents display a typical transient time course with a sigmoidal rise to a peak followed by an exponential decay. The rates of early activation and subsequent inactivation accelerate and approach a maximum in response to test potentials, V , of greater depolarization. The magnitude of peak sodium current increased from negligible values below V = −50 mV and reached a maximum at V = −3.6 mV ± 2.7 mV (mean ± S.E.M., n = 12). Sodium currents reversed at V = + 70 mV, near the predicted Nernst equilibrium potential for a Na + selective channel. The peak sodium conductance, g peak increased with depolarizing voltages to a maximum at V = ∼ 0 mV, exhibiting half-activation voltage at V ≽ −36.8 mV and an e -fold change in g peak /9.5 mV. The Hodgkin-Huxley inactivation parameter h ∞ indicates that at V = −73.6 mV half of the sodium currents were inactivated. Single channel current recordings demonstrated the occurrence of discrete events: the latency for first opening was shorter as the depolarizing pulse became more positive. The single-channel current amplitude was ohmic with a slope conductance, γ = 17.13 pS ± 0.66 pS. Sodium channel currents were reversibly blocked by tetrodotoxin (TTX). Thus, the TE671 human neurons in culture express a sodium channel which exhibits the voltage-gating properties, cation selectivity and neurotoxin sensitivity characteristic of ‘classical’ sodium channels of excitable Cells.

  • voltage gated sodium channels expressed in the human cerebellar Medulloblastoma Cell Line te671
    Brain Research, 1990
    Co-Authors: Franco Gambale, Mauricio Montal
    Abstract:

    A characterization of the properties of voltage-gated sodium channels expressed in the human cerebellar Medulloblastoma Cell Line TE671 is presented. Membrane currents were recorded under voltage clamp conditions using the patch clamp technique in both the whole-Cell and the excised-patch configurations. Macroscopic sodium currents display a typical transient time course with a sigmoidal rise to a peak followed by an exponential decay

Gert Lubec - One of the best experts on this subject based on the ideXlab platform.

  • identification of c myc dependent proteins in the Medulloblastoma Cell Line d425med
    Amino Acids, 2012
    Co-Authors: Amedeo A Azizi, Irene Slavc, Thomas Strobel, Weiqiang Chen, Lin Li, Gert Lubec
    Abstract:

    High c-myc levels are linked to poor prognosis in Medulloblastoma (MB), and it was the aim of the current study to search for c-myc-dependent proteins in the MB Cell Line D425Med. For this purpose D425Med Cells and Cells with knocked-down c-myc (by siRNA) were analysed by a gel-based differential proteomics study using mass spectrometry. Heterogeneous nuclear ribonucleoproteins C1/C2, heterogeneous nuclear ribonucleoprotein A/B, stathmin, endoplasmic reticulum protein ERp29 precursor and guanidinoacetate N-methyltransferase were c-myc dependently expressed. Signalling, the protein machinery, metabolism and endoplasmic reticulum function may be affected and these results enable studying tumour tissue for these proteins as potential dignity markers or pharmacological targets.

  • neurotrophin 3 trkc regulated proteins in the human Medulloblastoma Cell Line daoy
    Electrophoresis, 2009
    Co-Authors: Mariella Gruberolipitz, Irene Slavc, Thomas Strobel, Michael A Grotzer, Sung Ung Kang, Julius Paul Pradeep John, Gert Lubec
    Abstract:

    Medulloblastoma (MB) is the most common malignant childhood brain tumor and high neurotrophin (NP) receptor TrkC mRNA expression was identified as a powerful independent predictor of favorable survival outcome. In order to determine downstream effector proteins of TrkC signaling, the MB Cell Line DAOY was stably transfected with a vector containing the full-length TrkC cDNA sequence or an empty vector control. A proteomic approach was used to search for expressional changes by two mass spectrometric methods and immunoblotting for validation of significant results. Multiple time points for up to 48 h following NP-3-induced TrkC receptor activation were chosen. Thirteen proteins from several pathways (nucleoside diphosphate kinase A, stathmin, valosin-containing protein, annexin A1, dihydropyrimidinase-related protein-3, DJ-1 protein, glutathione S-transferase P, lamin A/C, fascin, cofilin, vimentin, vinculin, and moesin) were differentially expressed and most have been shown to play a role in differentiation, migration, invasion, proliferation, apoptosis, drug resistance, or oncogenesis. Knowledge on effectors of TrkC signaling may represent a first useful step for the identification of marker candidates or reflecting probable pharmacological targets for specific treatment of MB.

  • synthesis chaperoning and metabolism of proteins are regulated by nt 3 trkc signaling in the Medulloblastoma Cell Line daoy
    Journal of Proteome Research, 2008
    Co-Authors: Mariella Gruberolipitz, Irene Slavc, Thomas Strobel, Weiqiang Chen, Michael A Grotzer, Franz Quehenberger, Gert Lubec
    Abstract:

    The human Medulloblastoma Cell Line DAOY was transfected with Tropomyosin receptor kinase (TrkC), a marker for good prognostic outcome. Following TrkC-activation by its ligand neurotrophin-3, protein extracts from DAOY Cells were run on 2DE with subsequent MALDI-TOF-TOF analysis and quantification in order to detect downstream effectors. Protein levels of translational, splicing, processing, chaperone, protein handling, and metabolism machineries were shown to depend on neurotrophin-3-induced TrkC activation probably representing pharmacological targets.

  • mass spectrometric identification of serine hydrolase ovca2 in the Medulloblastoma Cell Line daoy
    Cancer Letters, 2006
    Co-Authors: Amedeo A Azizi, Jaewon Yang, Irene Slavc, Ellen Gelpi, Bernhard Rupp, Andrew K Godwin, Carolyn M Slater, Gert Lubec
    Abstract:

    Abstract OVCA2 is a putative serine-hydrolase. Performing protein profiling in human tumour Cell Lines, OVCA2 was detected in DAOY Medulloblastoma Cells as a high abundance protein. The protein was unambiguously identified by 2D gel-electrophoresis and MALDI-MS and MS/MS, its presence was confirmed by western blotting. Immunohistochemistry revealed expression in Medulloblastoma and predominantly in oligodendrocytes. Computational approaches predicted functional motifs and domains, interaction with apoptosis-related protein BAG and 3D structure. In addition to the presence of OVCA2 in Medulloblastoma, it was furthermore detectable in three out of 10 human tumour Cell-Lines as a high abundance protein probably suggesting a role in the tumour biology.

  • the Medulloblastoma Cell Line daoy but not eleven other tumor Cell Lines expresses minichromosome maintenance protein 4
    Cancer Letters, 2006
    Co-Authors: Mariella Gruberolipitz, Jaewon Yang, Thomas Stroebel, Irene Slavc, Gert Lubec
    Abstract:

    Minichromosome maintenance proteins (MCM) are required for initiation and elongation of chromosomal DNA, ensuring that DNA replication takes place only once. Although MCMs are considered of utmost importance in tumor biology and as potential marker proteins, they were not unambiguously identified at the protein level and we therefore aimed to characterize MCM 4 in a Medulloblastoma Cell Line and provide a protein chemical analytical tool. In addition, we searched for this protein in twelve tumor Cell Lines and a series of non-tumor Cells. The DAOY Medulloblastoma Cell Line was cultivated, lysed, proteins extracted and run on two-dimensional gel electrophoresis with subsequent in-gel digestion and mass spectrometrical (MS/MS) analysis of protein spots. One spot at pI 6.2 with an observed molecular weight of 98 kDa was identified as minichromosome maintenance protein 4 by peptide fingerprinting. Sequence coverage of 35% along with 25 matched peptides and MS/MS analyses of three matching peptides warranted unambiguous identification. The use of mass spectrometrical identification unequivocally allowed determination of MCM 4 expression in a Medulloblastoma Cell Line exclusively. Given the biological and probable clinical importance of this molecule as a tentative marker protein, a fair analytical tool, independent of antibody availability and specificity is mandatory and determinations at the transcriptional level cannot be extrapolated to protein levels per se, as there is a long and unpredictable way from mRNA to protein.

Kyu-chang Wang - One of the best experts on this subject based on the ideXlab platform.

  • Growth-inhibitory effect of neurotrophin-3-secreting adipose tissue-derived mesenchymal stem Cells on the D283-MED human Medulloblastoma Cell Line
    Journal of Neuro-Oncology, 2012
    Co-Authors: Seung Ah Choi, Kyu-chang Wang
    Abstract:

    Medulloblastoma (MBL), the most common malignant pediatric brain tumor, is incurable in about one-third of patients and can lead to long-term disabilities despite current multimodal treatments. The purpose of this study was to demonstrate in vitro biological effects of neurotrophins-3 (NT-3) on MBL Cells and to evaluate the growth-inhibitory effect of neurotrophin-3 (NT-3)-secreting stem Cells on tumor Cells. We confirmed by western blotting that D283-MED Cells express tyrosine kinase C, a specific receptor for NT-3. Analyzing the biological effects of NT-3 on MBL Cells, we evaluated autophagy, apoptosis, senescence, and differentiation of tumor Cells with NT-3. The NT-3 induced a concentration-dependent increase in apoptosis in the tumor Cell Line ( P  

  • growth inhibitory effect of neurotrophin 3 secreting adipose tissue derived mesenchymal stem Cells on the d283 med human Medulloblastoma Cell Line
    Journal of Neuro-oncology, 2012
    Co-Authors: Seung Ah Choi, Kyu-chang Wang
    Abstract:

    Medulloblastoma (MBL), the most common malignant pediatric brain tumor, is incurable in about one-third of patients and can lead to long-term disabilities despite current multimodal treatments. The purpose of this study was to demonstrate in vitro biological effects of neurotrophins-3 (NT-3) on MBL Cells and to evaluate the growth-inhibitory effect of neurotrophin-3 (NT-3)-secreting stem Cells on tumor Cells. We confirmed by western blotting that D283-MED Cells express tyrosine kinase C, a specific receptor for NT-3. Analyzing the biological effects of NT-3 on MBL Cells, we evaluated autophagy, apoptosis, senescence, and differentiation of tumor Cells with NT-3. The NT-3 induced a concentration-dependent increase in apoptosis in the tumor Cell Line (P < 0.001). The high concentrations of NT-3 increased the expression of class III β-tubulin (P < 0.001) and decreased the expression of Nestin (P < 0.05). NT-3-secreting stem Cells were produced by nucleofecting pIRES2.EGFP-NT3 into human adipose tissue-derived mesenchymal stem Cells (hAT-MSCs) and their tropic property toward MBL Cells was confirmed by migration assay. Double-layered co-culture experiments with the NT-3-secreting hAT-MSCs and D283-MED MBL Cells were performed, and NT-3-induced Cell death was studied by 3-(4,5-dimethylathiazol-2-yl)-2,5-dephenyl-tetrazolium bromide (MTT) assay. Consequently, the high concentrations of NT-3-secreting hAT-MSCs significantly (P < 0.05) increased the death of D283-MED Cells in vitro. The present study demonstrated that both apoptotic Cell death and neuronal differentiation of tumor Cells were the mechanisms of growth-inhibitory effect of NT-3-secreting hAT-MSCs on MBL Cell Line.

  • abstract 3450 growth inhibitory effect of neurotrophin 3 secreting adipose tissue derived mesenchymal stem Cells on the d283 med human Medulloblastoma Cell Line
    Cancer Research, 2011
    Co-Authors: Kyu-chang Wang, Seung Ah Choi, Chulkee Park
    Abstract:

    Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Medulloblastoma (MBL), the most common malignant pediatric brain tumor, is incurable in about one-third of patients and can lead to long-term disabilities despite current multimodal treatments. We evaluate the in vitro, growth-inhibitory effect of neurotrophin-3 (NT-3)-secreting stem Cells on an MBL Cell Line and propose that this Cell therapy may have therapeutic effects for MBL. NT-3-secreting stem Cells were produced by nucleofecting pIRES2.EGFP-NT3 into human adipose tissue-derived mesenchymal stem Cells (hAT-MSCs). Double-layered co-culture experiments with the NT-3-secreting hAT-MSCs and D283-MED MBL Cells were performed, and NT-3-induced apoptosis was studied by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) test. The growth-inhibitory effects of these stem Cells on human MBL in vitro were confirmed by 3-(4, 5-dimethylathiazol-2-yl)-2, 5-dephenyl-tetrazolium bromide (MTT) assay. We confirmed by western blotting that D283-MED Cells express tyrosine kinase C, a specific receptor for NT-3, and we showed by fluorescence microscopy and RT-PCR that transfected hAT-MSCs express NT-3. The double-layered co-culture of D283-MED with NT-3-secreting hAT-MSCs induced a concentration-dependent increase in apoptosis in the tumor Cell Line. Consequently, the high concentrations of NT-3-secreting hAT-MSCs significantly (P < 0.001) increased the death of D283-MED Cells in vitro. The present studies demonstrate the potential of NT-3-secreting hAT-MSCs as an effective therapeutic Cell therapy for human MBL in vitro, but in vivo experiments and clinical studies will be required in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3450. doi:10.1158/1538-7445.AM2011-3450

Corrado Carignani - One of the best experts on this subject based on the ideXlab platform.

  • inhibition of sk3 channels in the te671 human Medulloblastoma Cell Line by desipramine and imipramine
    European Journal of Pharmacology, 2002
    Co-Authors: Corrado Carignani, Mauro Corsi
    Abstract:

    Abstract The TE671 human Medulloblastoma Cell Line endogenously expresses SK3 channels. Using patch clamp, we tested the effects on this current of desipramine and imipramine. In both cases, we observed a complete, reversible and concentration-dependent block. The interaction of desipramine with the selective SK3 blocker, apamin, was studied in more detail. Co-application of desipramine and apamin at concentrations close to their IC 50 produced an additive effect that was significantly higher than that of each compound alone. This effect was also observed at IC 25 concentrations. Collectively, these data provide evidence against a common site of action for desipramine and apamin.

  • Pharmacological and molecular characterisation of SK3 channels in the TE671 human Medulloblastoma Cell Line.
    Brain research, 2002
    Co-Authors: Corrado Carignani, Renza Roncarati, Rebecca Rimini, Georg C Terstappen
    Abstract:

    The expression of the small conductance calcium-activated potassium channels SK1, SK2 and SK3 was investigated in the TE671 human Medulloblastoma Cell Line using RT-PCR and transcripts were detected only for SK3. Immunodetection experiments confirmed this result, demonstrating the presence of the SK3 protein. This potassium channel was characterised in TE671 Cells using whole-Cell patch-clamp recordings. Voltage steps to -100 mV from a holding potential of 0 mV in equimolar 140 mM intra- and extraCellular K(+) (K(+)(in/out)) elicited an inward current. The reversal potential of this current shifted 56.6 mV per 10-fold increase in K(+)(out) thus suggesting K(+) selectivity. This current was dependent on the concentration of Ca(2+)(in) with an EC(50) of 104.2 nM. A pharmacological characterisation of this current revealed that it was not blocked by 1 microM charybdotoxin (ChTX), 0.3 microM iberiotoxin (IbTX) or 10 microM clotrimazole (CLT) and only modestly inhibited (

  • pharmacological and molecular characterisation of sk3 channels in the te671 human Medulloblastoma Cell Line
    Brain Research, 2002
    Co-Authors: Corrado Carignani, Renza Roncarati, Rebecca Rimini, Georg C Terstappen
    Abstract:

    The expression of the small conductance calcium-activated potassium channels SK1, SK2 and SK3 was investigated in the TE671 human Medulloblastoma Cell Line using RT-PCR and transcripts were detected only for SK3. Immunodetection experiments confirmed this result, demonstrating the presence of the SK3 protein. This potassium channel was characterised in TE671 Cells using whole-Cell patch-clamp recordings. Voltage steps to −100 mV from a holding potential of 0 mV in equimolar 140 mM intra- and extraCellular K+ (K+in/out) elicited an inward current. The reversal potential of this current shifted 56.6 mV per 10-fold increase in K+out thus suggesting K+ selectivity. This current was dependent on the concentration of Ca2+in with an EC50 of 104.2 nM. A pharmacological characterisation of this current revealed that it was not blocked by 1 μM charybdotoxin (ChTX), 0.3 μM iberiotoxin (IbTX) or 10 μM clotrimazole (CLT) and only modestly inhibited (<50%) by 30 nM scyllatoxin (ScTX), 200 μM dequalinium chloride (Deq) or 300 μM d-tubocurarine (d-TC). The non-selective SK blocker d-TC blocked the current with an IC50 of 43.2 μM while apamin blocked the current to a much greater extent (87.8% at 1 μM) with an IC50 of 4.3 nM. Furthermore, the current was significantly increased (132.6±5.2%, n=7) by 500 μM 1-ethyl-2-benzimidazolinone (EBIO). Collectively, these data demonstrate the presence of an endogenous SK3 channel in human TE671 Cells.

Franco Gambale - One of the best experts on this subject based on the ideXlab platform.

  • voltage gated sodium channels expressed in the human cerebellar Medulloblastoma Cell Line te671
    Molecular Brain Research, 1990
    Co-Authors: Franco Gambale, Mauricio Montal
    Abstract:

    Abstract A characterization of the properties of voltage-gated sodium channels expressed in the human cerebellar Medulloblastoma Cell Line TE671 is presented. Membrane currents were recorded under voltage clamp conditions using the patch clamp technique in both the whole-Cell and the excised-patch configurations. Macroscopic sodium currents display a typical transient time course with a sigmoidal rise to a peak followed by an exponential decay. The rates of early activation and subsequent inactivation accelerate and approach a maximum in response to test potentials, V , of greater depolarization. The magnitude of peak sodium current increased from negligible values below V = −50 mV and reached a maximum at V = −3.6 mV ± 2.7 mV (mean ± S.E.M., n = 12). Sodium currents reversed at V = + 70 mV, near the predicted Nernst equilibrium potential for a Na + selective channel. The peak sodium conductance, g peak increased with depolarizing voltages to a maximum at V = ∼ 0 mV, exhibiting half-activation voltage at V ≽ −36.8 mV and an e -fold change in g peak /9.5 mV. The Hodgkin-Huxley inactivation parameter h ∞ indicates that at V = −73.6 mV half of the sodium currents were inactivated. Single channel current recordings demonstrated the occurrence of discrete events: the latency for first opening was shorter as the depolarizing pulse became more positive. The single-channel current amplitude was ohmic with a slope conductance, γ = 17.13 pS ± 0.66 pS. Sodium channel currents were reversibly blocked by tetrodotoxin (TTX). Thus, the TE671 human neurons in culture express a sodium channel which exhibits the voltage-gating properties, cation selectivity and neurotoxin sensitivity characteristic of ‘classical’ sodium channels of excitable Cells.

  • voltage gated sodium channels expressed in the human cerebellar Medulloblastoma Cell Line te671
    Brain Research, 1990
    Co-Authors: Franco Gambale, Mauricio Montal
    Abstract:

    A characterization of the properties of voltage-gated sodium channels expressed in the human cerebellar Medulloblastoma Cell Line TE671 is presented. Membrane currents were recorded under voltage clamp conditions using the patch clamp technique in both the whole-Cell and the excised-patch configurations. Macroscopic sodium currents display a typical transient time course with a sigmoidal rise to a peak followed by an exponential decay