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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 45 (1985), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Insulin receptors were detected in a variety of rat neuroblastoma and glioma cell lines. The binding of 125I-insulin to B103 neuroblastoma cells had characteristics typical of insulin receptors in other tissues, including high affinity for insulin, low affinity for insulin-like growth factor I (IGF-I), and curvilinear Scatchard plots. Using photoaffinity labeling procedures and sodium dodecyl sulfate (SDS) gel electrophoresis to analyze the subunit structure of insulin receptors in B103 cells, the predominantly labeled protein had an apparent molecular weight of 125K and the mobility of this protein was shifted after removal of sialic acid residues. On the basis of size and susceptibility to neuraminidase, the insulin binding subunit in neuroblastoma cells was identical to the α-subunit of insulin receptors in adipocytes and different from the 115K subunit found in brain. The presence of an “adipocyte′’ form of the insulin receptor in clonal cells derived from brain is probably a consequence of transformation and results from more extensive oligosaccharide processing of the 115K receptor expressed in normal brain cells. The fully glycosylated receptors in neuroblastoma cells were capable of exerting functions typical of insulin receptors in adipocytes such as internalization of insulin and stimulation of glucose transport.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 51 (1988), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: In this report, we have examined the structure, regulation, and function of insulin receptors in cultured neurons from fetal chicken brain. The apparent molecular weight of the α-subunit of neuronal insulin receptors, analyzed by photoaffinity labeling and sodium dodecyl sulfate gel electrophoresis under reducing conditions, was 115,000. The number of insulin receptors in the cultures increased from day 2 to day 4 during a period of extensive process formation. After 5 days in culture, there were approximately 40,000 high-affinity insulin receptors per neuron. When neurons were photoaffinity labeled at 16°C and then warmed to 37°C for 30 min, approximately 40% of the cell-surface receptors were recovered in the intracellular, trypsin-insensitive pool. Chronic exposure of neurons to insulin (100 ng/ml) resulted in a time-dependent loss of neuronal insulin receptors with a maximal decrease of 50% after 24 h. Insulin had no effect on glucose transport, glucose oxidation, or glycogen synthase activity in neurons. On the other hand, insulin supported the growth and differentiation of a fraction of neurons isolated from chick forebrain. We conclude that (1) cultured neurons from fetal chicken brain express the same subtype of insulin receptor previously identified in adult rat and human brain, (2) the neuronal subtype of insulin receptor undergoes internalization and down-regulation in response to insulin, and (3) neuronal insulin receptors do not acutely regulate glucose metabolism but mediate growth in neurons.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Primary cerebellar granule neurons (CGNs) require depolarizing extracellular potassium for their survival. Removal of depolarizing potassium triggers CGN apoptosis that requires induction of Bim, a BH3-only Bcl-2 family member. Bim is classically thought to promote apoptosis by neutralizing pro-survival Bcl-2 proteins. To determine if this is the principal function of Bim in CGNs, we contrasted Bim-mediated apoptosis to neuronal death induced by HA14-1, a BH3-domain mimetic that antagonizes Bcl-2 and Bcl-x(L). HA14-1 elicited CGN apoptosis characterized by caspase 3 and 9 activation, cytochrome c release, conformational activation of Bax, and mitochondrial depolarization. HA14-1 provoked CGN apoptosis in the absence of Bim induction and negative regulators of Bim transcription did not prevent HA14-1-induced cell death. However, the antioxidant glutathione and its precursor, N-acetyl-l-cysteine, suppressed HA14-1-induced apoptosis. Similarly, apoptosis induced by either a structurally distinct Bcl-2/Bcl-x(L) inhibitor (compound 6) or Bcl-2 antisense oligonucleotides was diminished by glutathione. In contrast, antioxidants had no effect on CGN apoptosis provoked by either removal of depolarizing potassium or overexpression of a GFP–Bim fusion protein, two models of Bim-dependent death. These data show that antagonism of Bcl-2/Bcl-x(L) function elicits oxidative stress-dependent CGN apoptosis that is mechanistically distinct from Bim-mediated cell death. These results further indicate that, although Bcl-2/Bcl-x(L) antagonism is sufficient to induce neuronal apoptosis, Bim likely promotes neuronal death by interacting with additional proteins besides Bcl-2/Bcl-x(L).
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Rho GTPases are key transducers of integrin/extracellular matrix and growth factor signaling. Although integrin-mediated adhesion and trophic support suppress neuronal apoptosis, the role of Rho GTPases in neuronal survival is unclear. Here, we have identified Rac as a critical pro-survival GTPase in cerebellar granule neurons (CGNs) and elucidated a death pathway triggered by its inactivation. GTP-loading of Rac1 was maintained in CGNs by integrin-mediated (RGD-dependent) cell attachment and trophic support. Clostridium difficile toxin B (ToxB), a specific Rho family inhibitor, induced a selective caspase-mediated degradation of Rac1 without affecting RhoA or Cdc42 protein levels. Both ToxB and dominant–negative N17Rac1 elicited CGN apoptosis, characterized by cytochrome c release and activation of caspase-9 and -3, whereas dominant–negative N19RhoA or N17Cdc42 did not cause significant cell death. ToxB stimulated mitochondrial translocation and conformational activation of Bax, c-Jun activation, and induction of the BH3-only protein Bim. Similarly, c-Jun activation and Bim induction were observed with N17Rac1. A c-jun N-terminal protein kinase (JNK)/p38 inhibitor, SB203580, and a JNK-specific inhibitor, SP600125, significantly decreased ToxB-induced Bim expression and blunted each subsequent step of the apoptotic cascade. These results indicate that Rac acts downstream of integrins and growth factors to promote neuronal survival by repressing c-Jun/Bim-mediated mitochondrial apoptosis.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: In vivo, the pesticide rotenone induces degeneration of dopamine neurons and parkinsonian-like pathology in adult rats. In the current study, we utilized primary ventral mesencephalic (VM) cultures from E15 rats as an in vitro model to examine the mechanism underlying rotenone-induced death of dopamine neurons. After 11 h of exposure to 30 nm rotenone, the number of dopamine neurons identified by tyrosine hydroxylase (TH) immunostaining declined rapidly with only 23% of the neurons surviving. By contrast, 73% of total cells survived rotenone treatment, indicating that TH+ neurons are more sensitive to rotenone. Examination of the role of apoptosis in TH+ neuron death, revealed that 10 and 30 nm rotenone significantly increased the number of apoptotic TH+ neurons from 7% under control conditions to 38 and 55%, respectively. The increase in apoptotic TH+ neurons correlated with an increase in immunoreactivity for active caspase-3 in TH+ neurons. The caspase-3 inhibitor, DEVD, rescued a significant number of TH+ neurons from rotenone-induced death. Furthermore, this protective effect lasted for at least 32 h post-rotenone and DEVD exposure, indicating lasting neuroprotection achieved with an intervention prior to the death commitment point. Our results show for the first time in primary dopamine neurons that, at low nanomolar concentrations, rotenone induces caspase-3-mediated apoptosis. Understanding the mechanism of rotenone-induced apoptosis in dopamine neurons may contribute to the development of new neuroprotective strategies against Parkinson's disease.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Annals of the New York Academy of Sciences 692 (1993), S. 0 
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Depolarization promotes the survival of cerebellar granule neurons via activation of the transcription factor myocyte enhancer factor 2D (MEF2D). Removal of depolarization induces hyperphosphorylation of MEF2D on serine/threonine residues, resulting in its decreased DNA binding and susceptibility to caspases. The subsequent loss of MEF2-dependent gene transcription contributes to the apoptosis of granule neurons. The kinase(s) that phosphorylates MEF2D during apoptosis is currently unknown. The serine/threonine kinase, glycogen synthase kinase-3β (GSK-3β), plays a pro-apoptotic role in granule neurons. To investigate a potential role for GSK-3β in MEF2D phosphorylation, we examined the effects of lithium, a non-competitive inhibitor of GSK-3β, on MEF2D activity in cultured cerebellar granule neurons. Lithium inhibited caspase-3 activation and chromatin condensation in granule neurons induced to undergo apoptosis by removal of depolarizing potassium and serum. Concurrently, lithium suppressed the hyperphosphorylation and caspase-mediated degradation of MEF2D. Moreover, lithium sustained MEF2 DNA binding and transcriptional activity in the absence of depolarization. Lithium also attenuated MEF2D hyperphosphorylation and apoptosis induced by calcineurin inhibition under depolarizing conditions, a GSK-3β-independent model of neuronal death. In contrast to lithium, MEF2D hyperphosphorylation was not inhibited by forskolin, insulin-like growth factor-I, or valproate, three mechanistically distinct inhibitors of GSK-3β. These results demonstrate that the kinase that phosphorylates and inhibits the pro-survival function of MEF2D in cerebellar granule neurons is a novel lithium target distinct from GSK-3β.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 144 (1990), S. 538-545 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In this study, we examined the effects of pertussis toxin (PT) on the ADP-ribosylation of guanine nucleotide binding proteins (G-proteins) and various insulinstimulated processes in cultured BC3H-1 myocytes. Treatment of intact myocytes with 0.1 μg/ml PT for 24 hours resulted in the complete ribosylation of a 41 kDa protein. The 41 kDa PT substrate was immunoprecipitated with antibodies directed against a synthetic peptide corresponding to a unique sequence in the alpha subunit of Gi-proteins. PT treatment of intact cells had no effect on insulin receptor binding or internalization. However, PT inhibited insulin-stimulated glucose transport at all insulin-concentrations tested (1-100ng/ml). Maximally stimulated glucose transport was reduced by 50% ± 15%. Insulin-stimulated glucose oxidation was also decreased by 31% ± 8%. The toxin had no significant effect on the basal rates of glucose transport and glucose oxidation. The time course of PT-induced inhibition on glucose transport correlated with the time course of the “in vivo” ADP-ribosylation of the 41 kDa protein. The results suggest that a 41 kDa PT-sensitive G-protein, identical or very similar to Gi, is involved in the regulation of glucose metabolism by insulin in BC3H-1 cells.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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