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Poly(Dimethylsiloxane) (PDMS) Affects Gene Expression in PC12 Cells Differentiating into Neuronal-Like Cells  [PDF]
Joanna M. ?opacińska, Jenny Emnéus, Martin Dufva
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0053107
Abstract: Introduction Microfluidics systems usually consist of materials like PMMA - poly(methyl methacrylate) and PDMS - poly(dimethylsiloxane) and not polystyrene (PS), which is usually used for cell culture. Cellular and molecular responses in cells grown on PS are well characterized due to decades of accumulated research. In contrast, the experience base is limited for materials used in microfludics chip fabrication. Methods The effect of different materials (PS, PMMA and perforated PMMA with a piece of PDMS underneath) on the growth and differentiation of PC12 (adrenal phaeochromocytoma) cells into neuronal-like cells was investigated using cell viability, cell cycle distribution, morphology, and gene expression analysis. Results/Conclusions After differentiation, the morphology, viability and cell cycle distribution of PC12 cells grown on PS, PMMA with and without PDMS underneath was the same. By contrast, 41 genes showed different expression for PC12 cells differentiating on PMMA as compared to on PS. In contrast, 677 genes showed different expression on PMMA with PDMS underneath as compared with PC12 cells on PS. The differentially expressed genes are involved in neuronal cell development and function. However, there were also many markers for neuronal cell development and functions that were expressed similarly in cells differentiating on PS, PMMA and PMMA with PDMS underneath. In conclusion, it was shown that PMMA has a minor impact and PDMS a major impact on gene expression in PC12 cells.
Chronic ethanol exposure increases microtubule content in PC12 cells
Cindy K Reiter-Funk, Douglas P Dohrman
BMC Neuroscience , 2005, DOI: 10.1186/1471-2202-6-16
Abstract: Chronic ethanol exposure of wild-type and vector control PC12 cells resulted in a significant increase in microtubule content and a corresponding decrease in free tubulin. There was also a significant increase in microtubule content in PC12 cells expressing a dominate-negative inhibitor of epsilon PKC; cells which have previously been shown to have no ethanol-induced increase in neurite outgrowth. In contrast, ethanol had no effect on microtubule content in PC12 cells expressing a dominate-negative inhibitor of delta PKC.These results suggest that chronic ethanol exposure alters the relative ratio of free tubulin to microtubule-associated tubulin, an important component of the cytoskeleton. Further, the data from the PKC dominant-negative cell lines suggest that the effects of ethanol on microtubule content do not correlate with the effects of ethanol on neurite outgrowth. The delta isoform of PKC appears to be necessary for the ethanol-induced increase in microtubule content. These studies demonstrate an effect of chronic ethanol exposure which may contribute to previously documented alterations of neuronal cyto-architecture.Chronic ethanol exposure has been shown to cause damage to the adult and developing nervous system [1,2]. For example, in vivo chronic ethanol has been shown to cause aberrant sprouting of hippocampal neurites in developing rats [3], increase the length of dendrites in cerebellar Purkinje neurons [4], the size of synaptic terminals of cerebellar granule cells [5], and the number of dendritic spines on hippocampal dentate granule neurons in adult rats [6]. Furthermore, in vitro ethanol enhances neurite outgrowth in cultured rat cerebellar neurons [7]. Contrary to the enhancement of neurite outgrowth, other studies have shown that chronic ethanol exposure inhibits the growth of dendrites in CA1 hippocampal neurons and cerebellar Purkinje cells in vivo and inhibits chick spinal cord neurite formation in vitro [8,9]. However, the mechanisms underly
α-Tocopherol at Nanomolar Concentration Protects PC12 Cells from Hydrogen Peroxide-Induced Death and Modulates Protein Kinase Activities  [PDF]
Irina O. Zakharova,Tatyana V. Sokolova,Liubov V. Bayunova,Yulia A. Vlasova,Maria P. Rychkova,Natalia F. Avrova
International Journal of Molecular Sciences , 2012, DOI: 10.3390/ijms130911543
Abstract: The aim of this work was to compare protective and anti-apoptotic effects of α-tocopherol at nanomolar and micromolar concentrations against 0.2 mM H 2O 2-induced toxicity in the PC12 neuronal cell line and to reveal protein kinases that contribute to α-tocopherol protective action. The protection by 100 nM α-tocopherol against H 2O 2-induced PC12 cell death was pronounced if the time of pre-incubation with α-tocopherol was 3–18 h. For the first time, the protective effect of α-tocopherol was shown to depend on its concentration in the nanomolar range (1 nM < 10 nM < 100 nM), if the pre-incubation time was 18 h. Nanomolar and micromolar α-tocopherol decreased the number of PC12 cells in late apoptosis induced by H 2O 2 to the same extent if pre-incubation time was 18 h. Immunoblotting data showed that α-tocopherol markedly diminished the time of maximal activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and protein kinase B (Akt)-induced in PC12 cells by H 2O 2. Inhibitors of MEK 1/2, PI 3-kinase and protein kinase C (PKC) diminished the protective effect of α-tocopherol against H 2O 2-initiated toxicity if the pre-incubation time was long. The modulation of ERK 1/2, Akt and PKC activities appears to participate in the protection by α-tocopherol against H 2O 2-induced death of PC12 cells. The data obtained suggest that inhibition by α-tocopherol in late stage ERK 1/2 and Akt activation induced by H 2O 2 in PC12 cells makes contribution to its protective effect, while total inhibition of these enzymes is not protective.
Interplay between Cell Migration and Neurite Outgrowth Determines SH2B1β-Enhanced Neurite Regeneration of Differentiated PC12 Cells  [PDF]
Chia-Ling Wu, Yu-Han Chou, Yu-Jung Chang, Nan-Yuan Teng, Hsin-Ling Hsu, Linyi Chen
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0034999
Abstract: The regulation of neurite outgrowth is crucial in developing strategies to promote neurite regeneration after nerve injury and in degenerative diseases. In this study, we demonstrate that overexpression of an adaptor/scaffolding protein SH2B1β promotes neurite re-growth of differentiated PC12 cells, an established neuronal model, using wound healing (scraping) assays. Cell migration and the subsequent remodeling are crucial determinants during neurite regeneration. We provide evidence suggesting that overexpressing SH2B1β enhances protein kinase C (PKC)-dependent cell migration and phosphatidylinositol 3-kinase (PI3K)-AKT-, mitogen activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) kinase (MEK)-ERK-dependent neurite re-growth. Our results further reveal a cross-talk between pathways involving PKC and ERK1/2 in regulating neurite re-growth and cell migration. We conclude that temporal regulation of cell migration and neurite outgrowth by SH2B1β contributes to the enhanced regeneration of differentiated PC12 cells.
Luteolin Induces microRNA-132 Expression and Modulates Neurite Outgrowth in PC12 Cells  [PDF]
Lian-Fang Lin,Szu-Ping Chiu,Ming-Jiuan Wu,Pei-Yi Chen,Jui-Hung Yen
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0043304
Abstract: Luteolin (3′,4′,5,7-tetrahydroxyflavone), a food-derived flavonoid, has been reported to exert neurotrophic properties that are associated with its capacity to promote neuronal survival and neurite outgrowth. In this study, we report for the first time that luteolin induces the persistent expression of microRNA-132 (miR-132) in PC12 cells. The correlation between miR-132 knockdown and a decrease in luteolin-mediated neurite outgrowth may indicate a mechanistic link by which miR-132 functions as a mediator for neuritogenesis. Furthermore, we find that luteolin led to the phosphorylation and activation of cAMP response element binding protein (CREB), which is associated with the up-regulation of miR-132 and neurite outgrowth. Moreover, luteolin-induced CREB activation, miR-132 expression and neurite outgrowth were inhibited by adenylate cyclase, protein kinase A (PKA) and MAPK/ERK kinase 1/2 (MEK1/2) inhibitors but not by protein kinase C (PKC) or calcium/calmodulin-dependent protein kinase II (CaMK II) inhibitors. Consistently, we find that luteolin treatment increases ERK phosphorylation and PKA activity in PC12 cells. These results show that luteolin induces the up-regulation of miR-132, which serves as an important regulator for neurotrophic actions, mainly acting through the activation of cAMP/PKA- and ERK-dependent CREB signaling pathways in PC12 cells.
Neurotrophic Effect of Citrus 5-Hydroxy-3,6,7,8,3′,4′-Hexamethoxyflavone: Promotion of Neurite Outgrowth via cAMP/PKA/CREB Pathway in PC12 Cells  [PDF]
Hui-Chi Lai, Ming-Jiuan Wu, Pei-Yi Chen, Ting-Ting Sheu, Szu-Ping Chiu, Meng-Han Lin, Chi-Tang Ho, Jui-Hung Yen
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0028280
Abstract: 5-Hydroxy-3,6,7,8,3′,4′-hexamethoxyflavo?ne(5-OH-HxMF), a hydroxylated polymethoxyflavone, is found exclusively in the Citrus genus, particularly in the peels of sweet orange. In this research, we report the first investigation of the neurotrophic effects and mechanism of 5-OH-HxMF in PC12 pheochromocytoma cells. We found that 5-OH-HxMF can effectively induce PC12 neurite outgrowth accompanied with the expression of neuronal differentiation marker protein growth-associated protein-43(GAP-43). 5-OH-HxMF caused the enhancement of cyclic AMP response element binding protein (CREB) phosphorylation, c-fos gene expression and CRE-mediated transcription, which was inhibited by 2-naphthol AS-E phosphate (KG-501), a specific antagonist for the CREB-CBP complex formation. Moreover, 5-OH-HxMF-induced both CRE transcription activity and neurite outgrowth were inhibited by adenylate cyclase and protein kinase A (PKA) inhibitor, but not MEK1/2, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) or calcium/calmodulin-dependent protein kinase (CaMK) inhibitor. Consistently, 5-OH-HxMF treatment increased the intracellular cAMP level and downstream component, PKA activity. We also found that addition of K252a, a TrKA antagonist, significantly inhibited NGF- but not 5-OH-HxMF-induced neurite outgrowth. These results reveal for the first time that 5-OH-HxMF is an effective neurotrophic agent and its effect is mainly through a cAMP/PKA-dependent, but TrKA-independent, signaling pathway coupling with CRE-mediated gene transcription. A PKC-dependent and CREB-independent pathway was also involved in its neurotrophic action.
The Visual Orbit of iota Pegasi  [PDF]
A. F. Boden,C. D. Koresko
Physics , 1998, DOI: 10.1086/307030
Abstract: We have determined the visual orbit for the spectroscopic binary iota~Pegasi with interferometric visibility data obtained by the Palomar Testbed Interferometer in 1997. iota~Pegasi is a double-lined binary system whose minimum masses and spectral typing suggests the possibility of eclipses. Our orbital and component diameter determinations do not favor the eclipse hypothesis: the limb-to-limb separation of the two components is 0.151 +/- 0.069 mas at conjunction. Our conclusion that the iota~Peg system does not eclipse is supported by high-precision photometric observations. The physical parameters implied by our visual orbit and the spectroscopic orbit of Fekel and Tomkin (1983) are in good agreement with those inferred by other means. In particular, the orbital parallax of the system is determined to be 86.9 +/- 1.0 mas, and masses of the two components are determined to be 1.326 +/- 0.016 M_sun and 0.819 +/- 0.009 M_sun respectively.
Inhibition of Store-Operated Calcium Entry Attenuates MPP+-Induced Oxidative Stress via Preservation of Mitochondrial Function in PC12 Cells: Involvement of Homer1a  [PDF]
Xia Li, Wei Chen, Lei Zhang, Wen-bo Liu, Zhou Fei
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0083638
Abstract: The process of store-operated calcium entry (SOCE), whereby the release of intracellular Ca2+ from endoplasmic reticulum (ER) activates Ca2+ influx channels in the plasma membrane, has been demonstrated to impact a diverse range of cell functions. In the present study, we investigated the potential protective effect of SOCE inhibition against 1-methyl-4-phenylpyridinium (MPP+) injury by using pharmacological antagonists or specific small interfering RNA (siRNA) in PC12 cells. The results showed that both antagonists (15 μM MRS-1845 and 50 μM ML-9) and stromal interacting molecule-1 (STIM1) targeted siRNA (Si-STIM1) significantly increased cell viability, decreased apoptotic cell death and reduced intracellular reactive oxygen species (ROS) production and lipid peroxidation in MPP+ injured PC12 cells. SOCE inhibition also prevented MPP+ induced mitochondrial dysfunction and activation of mitochondrial related apoptotic factors, while had no effect on mitochondrial biogenesis. Moreover, inhibition of SOCE by antagonists and siRNA increased the expression levels of Homer1a mRNA and protein, and knockdown of Homer1a expression by specific siRNA partly reversed the protective effects induced by SOCE inhibition in PC12 cells. All these results indicated that SOCE inhibition protected PC12 cells against MPP+ insult through upregulation of Homer1a expression, and SOCE might be an ideal target for investigating therapeutic strategy against neuronal injury in PD patients.
Protein Kinase C Delta (PKCδ) Affects Proliferation of Insulin-Secreting Cells by Promoting Nuclear Extrusion of the Cell Cycle Inhibitor p21Cip1/WAF1  [PDF]
Felicia Ranta, Johannes Leveringhaus, Dorothea Theilig, Gabriele Schulz-Raffelt, Anita M. Hennige, Dominic G. Hildebrand, René Handrick, Verena Jendrossek, Fatima Bosch, Klaus Schulze-Osthoff, Hans-Ulrich H?ring, Susanne Ullrich
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0028828
Abstract: Background High fat diet-induced hyperglycemia and palmitate-stimulated apoptosis was prevented by specific inhibition of protein kinase C delta (PKCδ) in β-cells. To understand the role of PKCδ in more detail the impact of changes in PKCδ activity on proliferation and survival of insulin-secreting cells was analyzed under stress-free conditions. Methodology and Principal Findings Using genetic and pharmacological approaches, the effect of reduced and increased PKCδ activity on proliferation, apoptosis and cell cycle regulation of insulin secreting cells was examined. Proteins were analyzed by Western blotting and by confocal laser scanning microscopy. Increased expression of wild type PKCδ (PKCδWT) significantly stimulated proliferation of INS-1E cells with concomitant reduced expression and cytosolic retraction of the cell cycle inhibitor p21Cip1/WAF1. This nuclear extrusion was mediated by PKCδ-dependent phosphorylation of p21Cip1/WAF1 at Ser146. In kinase dead PKCδ (PKCδKN) overexpressing cells and after inhibition of endogenous PKCδ activity by rottlerin or RNA interference phosphorylation of p21Cip1/WAF1 was reduced, which favored its nuclear accumulation and apoptotic cell death of INS-1E cells. Human and mouse islet cells express p21Cip1/WAF1 with strong nuclear accumulation, while in islet cells of PKCδWT transgenic mice the inhibitor resides cytosolic. Conclusions and Significance These observations disclose PKCδ as negative regulator of p21Cip1/WAF1, which facilitates proliferation of insulin secreting cells under stress-free conditions and suggest that additional stress-induced changes push PKCδ into its known pro-apoptotic role.
Varicones and Growth Cones: Two Neurite Terminals in PC12 Cells  [PDF]
Ana Mingorance-Le Meur, Alma N. Mohebiany, Timothy P. O'Connor
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0004334
Abstract: The rat adrenal pheochromocytoma PC12 cell line is one of the traditional models for the study of neurite outgrowth and growth cone behavior. To clarify to what extent PC12 neurite terminals can be compared to neuronal growth cones, we have analyzed their morphology and protein distribution in fixed PC12 cells by immunocytochemistry. Our results show that that PC12 cells display a special kind of neurite terminal that includes a varicosity in close association with a growth cone. This hybrid terminal, or “varicone”, is characterized by the expression of specific markers not typically present in neuronal growth cones. For example, we show that calpain-2 is a specific marker of varicones and can be detected even before the neurite develops. Our data also shows that a fraction of PC12 neurites end in regular growth cones, which we have compared to hippocampal neurites as a control. We also report the extraordinary incidence of varicones in the literature referred to as “growth cones”. In summary, we provide evidence of two different kinds of neurite terminals in PC12 cells, including a PC12-specific terminal, which implies that care must be taken when using them as a model for neuronal growth cones or neurite outgrowth.
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