scholarly journals Purification of a dichlorophenol-indophenol oxidoreductase from rat and bovine synaptic membranes: tight complex association of a glyceraldehyde-3-phosphate dehydrogenase isoform, TOAD64, enolase-γ and aldolase C

1997 ◽  
Vol 324 (2) ◽  
pp. 555-563 ◽  
Author(s):  
Catherine BULLIARD ◽  
Rinaldo ZURBRIGGEN ◽  
Joelle TORNARE ◽  
Mahamadou FATY ◽  
Zubin DASTOOR ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cell Line ◽  
Plasma Membranes ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Synaptic Plasma Membranes ◽  
Rnase Protection ◽  
Intact Cells ◽  
Aldolase C ◽  
Sds Page

NADH–dichlorophenol-indophenol oxidoreductases (PMOs) were purified from synaptic plasma membranes or synaptic vesicles (small recycling vesicles) from both bovine and rat brains and from a neuroblastoma cell line, NB41A3. Several isoforms could be identified in purified plasma membranes and vesicles. Purification of the enzyme activity involved protein extraction with detergents, (NH4)2SO4 precipitation, chromatography under stringent conditions and native PAGE. PMO activity could be attributed to a very tight complex of several proteins that could not be separated except by SDS/PAGE. SDS/PAGE resolved the purified complex into at least five proteins, which could be micro-sequenced and identified unambiguously as hsc70, TOAD64 and glyceraldehyde-3-phosphate dehydrogenase tightly associated with the brain-specific proteins aldolase C and enolase-γ. Enzyme activity could be purified from both synaptic plasma membranes and recycling vesicles, yields being much greater from the latter source. Highly purified plasma membranes (prepared from a neuroblastoma cell line NB41A3 by iminobiotinylation of intact cells and affinity purification with avidin and anti-avidin antibodies under very stringent conditions) also displayed PMO activity tightly associated with TOAD64. The association of PMO in a tight complex was confirmed by its immunoprecipitation from cellular and membrane extracts of NB41A3 using antibodies directed against any component protein of the complex followed by immunodetection with antibodies directed against the other members. Antibodies also inhibited the enzyme activity synergistically. In addition, induction of the different components of the complex during dichlorophenol-indophenol stress was demonstrated by the S1 RNase-protection assay in synchronized NB41A3 cells. The role of the complex in membrane fusion and cellular response to extracellular oxidative stress during growth and development is discussed.

scholarly journals HMGB1 Receptors as Potential Novel Targets for Neuroblastoma

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Christian Garcia ◽  
Jared Smith ◽  
Peter Malicky ◽  
Fletcher White
Keyword(s):  
Cell Line ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
High Mobility ◽  
Neuroblastoma Cell Line ◽  
Receptor Protein ◽  
Cellular Mechanisms ◽  
Cxcr4 Receptor ◽  
Sds Page ◽  
Secondary Antibodies

Background and Hypothesis: The pathogenesis of neuroblastoma remains elusive. In order to further understand the pathogenesis, it is necessary to identify molecular and cellular mechanisms of the cancer. One protein of interest is high-mobility group box 1 protein (HMGB1). HMGB1 has been implicated in cancer including tumor growth, invasion, angiogenesis, metastasis, relapse and therapeutic resistance. HMGB1 isoforms signals via different receptors including the chemokine receptor, CXCR4, receptor for advanced glycation end products (RAGE), and the prominent inflammation-associated receptor, toll-like receptor 4 (TLR4). To gain insight into the possible impact of HMGB1 on the SH-SY5Y neuroblastoma cell line, we determined the degree to which CXCR4, TLR4 and RAGE are present on SH-SY5Y cells. We hypothesize that the presence of these receptors in SH-SY5Y cells may mediate proliferation of neuroblastoma cells and other types of cancer. Experimental Design or Project Methods: The SH-SY5Y cell line (ATCC® CRL-2266™) was derived from a metastatic bone tumor in a 4 year old female. Cells were lysed in lysis buffer, electrophoresed on a 10% SDS-PAGE, and blotted onto PVDF membrane. After blocking, the membranes were incubated with primary antibodies against the receptor protein overnight at 4 °C, and then with HRP-conjugated secondary antibodies for 1h. Protein bands were visualized with a SuperSignal West Pico Chemiluminescent Substrate. Results: To be finalized Conclusion and Potential Impact: Investigating SH-SY5Y cell line for the presence of these inflammation-associated receptors could potentially serve as a model to better understand the role of these receptors in cancer research.

scholarly journals Pyrido[2′,1′:2,3]imidazo[4,5-c]isoquinolin-5-amines as Potential Cytotoxic Agents against Human Neuroblastoma

2021 ◽  
Vol 14 (8) ◽  
pp. 750
Author(s):  
Zahira Tber ◽  
Mohammed Loubidi ◽  
Jabrane Jouha ◽  
Ismail Hdoufane ◽  
Mümin Alper Erdogan ◽  
...  
Keyword(s):  
Cell Line ◽  
Drug Exposure ◽  
Caspase 3 ◽  
Biological Activities ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Cytotoxic Agents ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma ◽  
Parp 1

We report herein the evaluation of various pyrido[2′,1′:2,3]imidazo[4,5-c]isoquinolin-5-amines as potential cytotoxic agents. These molecules were obtained by developing the multicomponent Groebke–Blackburn–Bienaymé reaction to yield various pyrido[2′,1′:2,3]imidazo[4,5-c]quinolines which are isosteres of ellipticine whose biological activities are well established. To evaluate the anticancer potential of these pyrido[2′,1′:2,3]imidazo[4,5-c]isoquinolin-5-amine derivatives in the human neuroblastoma cell line, the cytotoxicity was examined using the WST-1 assay after 72 h drug exposure. A clonogenic assay was used to assess the ability of treated cells to proliferate and form colonies. Protein expressions (Bax, bcl-2, cleaved caspase-3, cleaved PARP-1) were analyzed using Western blotting. The colony number decrease in cells was 50.54%, 37.88% and 27.12% following exposure to compounds 2d, 2g and 4b respectively at 10 μM. We also show that treating the neuroblastoma cell line with these compounds resulted in a significant alteration in caspase-3 and PARP-1 cleavage.

scholarly journals Divergent Pathways Regulate Ligand-Independent Activation of ERα in SK-N-BE Neuroblastoma and COS-1 Renal Carcinoma Cells

1998 ◽  
Vol 12 (6) ◽  
pp. 835-841
Author(s):  
Cesare Patrone ◽  
Elisabetta Gianazza ◽  
Sabrina Santagati ◽  
Paola Agrati ◽  
Adriana Maggi
Keyword(s):  
Cell Line ◽  
Intracellular Signaling ◽  
Steroid Receptors ◽  
Neuroblastoma Cell ◽  
Cross Coupling ◽  
Activation Function ◽  
Neuroblastoma Cell Line ◽  
Membrane Receptors ◽  
Insulin Dependent ◽  
The Cross

Abstract The α-estrogen receptor (ERα) transcriptional activity can be regulated either by binding to the cognate ligand or by intracellular signaling pathways responsive to a variety of factors acting through cell membrane receptors. Studies carried out in HeLa and COS-1 cells demonstrated that the cross-coupling between estrogen and growth factor receptors is mediated by p21ras and requires phosphorylation of a specific serine residue (Ser 118 in the human ERα and Ser 122 in mouse ERα) located in the ERα N-terminal activation function 1 (AF-1). Likewise, in the SK-N-BE neuroblastoma cell line p21ras is involved in the cross-coupling between insulin and ERα receptors. However, in this cell line Ser 122 is not necessary for insulin-dependent activation of unliganded ERα. In addition, after insulin activation, the electrophoretic mobility associated to serine hyperphosphorylation of ERα in SK-N-BE and in COS-1 cells is different. Our study rules out the possibility of tyrosine phosporylation in unliganded ERα activation by means of transactivation studies of ERα tyrosine mutants and analysis of Tyr phosphorylation immunoreactivity. The two cofactors for steroid receptors RIP 140 and SRC-1 do not seem to be specifically involved in the insulin-induced ERα transactivation. The present study demonstrates the possibility of an alternative, cell-specific pathway of cross-coupling between intracellular and membrane receptors, which might be of importance for the understanding of the physiological significance of this mode of activation in the nervous system.

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