Efficient genetic manipulation of 1321N1 astrocytoma cells using lentiviral gene transfer

2012 ◽  
Vol 206 (2) ◽  
pp. 138-142 ◽  
Author(s):  
Anja Keim ◽  
Isabelle Müller ◽  
Gerald Thiel
Keyword(s):  
Gene Transfer ◽  
Genetic Manipulation ◽  
Astrocytoma Cells ◽  
1321N1 Astrocytoma

scholarly journals Caveolin-1 Regulates P2Y2 Receptor Signaling during Mechanical Injury in Human 1321N1 Astrocytoma

Biomolecules ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 622
Author(s):  
Magdiel Martínez ◽  
Namyr A. Martínez ◽  
Jorge D. Miranda ◽  
Héctor M. Maldonado ◽  
Walter I. Silva Ortiz
Keyword(s):  
Cell Viability ◽  
Mechanical Injury ◽  
P2y2 Receptor ◽  
Caspase 9 ◽  
Post Injury ◽  
Caveolin 1 ◽  
Receptor Coupling ◽  
Downstream Signaling ◽  
Astrocytoma Cells ◽  
1321N1 Astrocytoma

Caveolae-associated protein caveolin-1 (Cav-1) plays key roles in cellular processes such as mechanosensing, receptor coupling to signaling pathways, cell growth, apoptosis, and cancer. In 1321N1 astrocytoma cells Cav-1 interacts with the P2Y2 receptor (P2Y2R) to modulate its downstream signaling. P2Y2R and its signaling machinery also mediate pro-survival actions after mechanical injury. This study determines if Cav-1 knockdown (KD) affects P2Y2R signaling and its pro-survival actions in the 1321N1 astrocytoma cells mechanical injury model system. KD of Cav-1 decreased its expression in 1321N1 cells devoid of or expressing hHAP2Y2R by ~88% and ~85%, respectively. Cav-1 KD had no significant impact on P2Y2R expression. Post-injury densitometric analysis of pERK1/2 and Akt activities in Cav-1-positive 1321N1 cells (devoid of or expressing a hHAP2Y2R) revealed a P2Y2R-dependent temporal increase in both kinases. These temporal increases in pERK1/2 and pAkt were significantly decreased in Cav-1 KD 1321N1 (devoid of or expressing a hHAP2Y2R). Cav-1 KD led to an ~2.0-fold and ~2.4-fold decrease in the magnitude of the hHAP2Y2R-mediated pERK1/2 and pAkt kinases’ activity, respectively. These early-onset hHAP2Y2R-mediated signaling responses in Cav-1-expressing and Cav-1 KD 1321N1 correlated with changes in cell viability (via a resazurin-based method) and apoptosis (via caspase-9 expression). In Cav-1-positive 1321N1 cells, expression of hHAP2Y2R led to a significant increase in cell viability and decreased apoptotic (caspase-9) activity after mechanical injury. In contrast, hHAP2Y2R-elicited changes in viability and apoptotic (caspase-9) activity were decreased after mechanical injury in Cav-1 KD 1321N1 cells expressing hHAP2Y2R. These findings support the importance of Cav-1 in modulating P2Y2R signaling during mechanical injury and its protective actions in a human astrocytoma cell line, whilst shedding light on potential new venues for brain injury or trauma interventions.

scholarly journals Genetic manipulation of primitive leukemic and normal hematopoietic cells using a novel method of adenovirus-mediated gene transfer

Leukemia ◽  
1999 ◽  
Vol 13 (10) ◽  
pp. 1608-1616 ◽  
Author(s):  
DS Howard ◽  
DA Rizzierri ◽  
B Grimes ◽  
D Upchurch ◽  
GL Phillips ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Genetic Manipulation ◽  
Hematopoietic Cells ◽  
Novel Method

Intramolecular Diels-Alder Cycloaddition: 7-Isocyanoamphilecta- 11(20),15-diene (Miyaoka), (–)-Scabronine G (Kanoh), Basiliolide B (Stoltz), Hirsutellone B (Uchiro), Echinopine A (Chen)

2015 ◽  
Author(s):  
Douglass F. Taber
Keyword(s):  
Neurotrophic Factors ◽  
Claisen Rearrangement ◽  
Diels Alder ◽  
Relative Configuration ◽  
Intramolecular Cycloaddition ◽  
Quaternary Centers ◽  
Astrocytoma Cells ◽  
National University ◽  
Seoul National University ◽  
1321N1 Astrocytoma

The amphilectane diterpenes, exemplified by 7-isocyanoamphilecta-11(20),15-diene 3, have been little investigated. In the course of a synthesis of 3, Hiroaki Miyaoka of the Tokyo University of Pharmacy and Life Sciences took advantage (Synlett 2011, 547) of the kinetic enolization and silylation of 1 to convert it into a trienone that spontaneously cyclized to 2. Scabronine G 6, isolated from the mushroom Sarcodon scabrosus, was found to enhance the secretion of neurotrophic factors from 1321N1 astrocytoma cells. To set the absolute configuration of the two quaternary centers that are 1, 4 on the cyclohexane ring of 6, Naoki Kanoh and Yoshiharu Iwabuchi of Tohoku University cyclized (Org. Lett. 2011, 13, 2864) 4 to 5. Although described by the authors as a double Michael addition, this transformation has the same connectivity as an intramolecular Diels-Alder cycloaddition. The diterpenes isolated from the genus Thapsia, represented by basiliolide B 9, induce rapid mobilization of intracellular Ca2+ stores. Brian M. Stoltz of Caltech effected (Angew. Chem. Int. Ed. 2011, 50, 3688) Claisen rearrangement of 7 to give an intermediate that cyclized to 8 as a mixture of diastereomers. A significant challenge in the synthesis was the assembly of the delicate enol ether/lactone of 9. Hirsutellone B 12, isolated from Hirsutella nivea, shows significant antituberculosis activity. Hiromi Uchiro of the Tokyo University of Science found it useful (Org. Lett. 2011, 13, 6268) to protect the intermediate unsaturated keto ester by intermolecular cycloaddition with pentamethylcyclopentadiene before constructing the triene of 10. Simple thermolysis reversed the intermolecular addition, opening the way to intramolecular cycloaddition to give 11. The tetracyclic ring system of the diterpene echinopine A 15 represents a substantial synthetic challenge. David Y.-K. Chen of Seoul National University approached this problem (Org. Lett. 2011, 13, 5724) by Pd-mediated cyclization of 13 to the diene, which then underwent intramolecular Diels-Alder cycloaddition to give 14, with control of the relative configuration of two of the three ternary centers of 15. Double bond migration followed by oxidative cleavage of the resulting cyclohexenone then set the stage for the intramolecular cyclopropanation that completed the synthesis of 15.

scholarly journals Thrombin receptors modulate insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate accumulation in 1321N1 astrocytoma cells

1996 ◽  
Vol 317 (2) ◽  
pp. 347-351 ◽  
Author(s):  
Ian H. BATTY ◽  
C. Peter DOWNES
Keyword(s):  
Insulin Receptors ◽  
Thrombin Receptor ◽  
Astrocytoma Cells ◽  
Thrombin Receptors ◽  
Transient Activation ◽  
Ligand Type ◽  
Insulin Receptor Signalling ◽  
Human Thrombin ◽  
Tethered Ligand ◽  
1321N1 Astrocytoma

Thrombin and insulin receptor signalling via phosphoinositide (PI)-specific phospholipase C (PLC) and PI 3-kinase was studied in [3H]inositol-labelled 1321N1 cells. Thrombin stimulated a dramatic, transient activation of PLC which is probably mediated via receptors of the ‘tethered-ligand’ type, since it was both reproduced by, and abolished following, pretreatment of cells with a synthetic peptide (SFLLRN) corresponding to the ligand domain of the human thrombin receptor. However, neither thrombin nor SFLLRN stimulated PI 3-kinase. By contrast, insulin did not influence [3H]InsP3 concentrations but stimulated accumulation of [3H]PtdIns(3,4,5)P3 and [3H]PtdIns(3,4)P2, the relative steady-state concentrations of which may indicate degradation of [3H]PtdIns(3,4,5)P3 by 5- and 3-phosphatases. The independent coupling of thrombin and insulin receptors to PLC and PI 3-kinase respectively in 1321N1 cells allowed interactions between these systems to be examined. Thus insulin-stimulated [3H]PtdIns(3,4,5)P3 accumulation was attenuated on co-stimulation of the thrombin receptor, whereas concentrations of [3H]PtdIns(3,4)P2 were transiently enhanced but then reduced. These results indicate that thrombin receptors in 1321N1 cells do not activate PI 3-kinase, but can modulate signalling by this enzyme.

THE POTENTIAL FOR GENETIC MANIPULATION OF PEACH FRUIT DEVELOPMENT AND QUALITY THROUGH DIRECTED GENE TRANSFER

1990 ◽  
pp. 503-508
Author(s):  
R. Scorza ◽  
P.H. Morgens ◽  
J.M. Cordts ◽  
R.A. Cohen ◽  
S. Mante ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Fruit Development ◽  
Genetic Manipulation ◽  
Peach Fruit

Ammonium-induced calcium mobilization in 1321N1 astrocytoma cells

2008 ◽  
Vol 227 (1) ◽  
pp. 36-47 ◽  
Author(s):  
Petra Hillmann ◽  
Meryem Köse ◽  
Kristina Söhl ◽  
Christa E. Müller
Keyword(s):  
Calcium Mobilization ◽  
Astrocytoma Cells ◽  
1321N1 Astrocytoma

scholarly journals Muscarinic-receptor-mediated inhibition of insulin-like growth factor-1 receptor-stimulated phosphoinositide 3-kinase signalling in 1321N1 astrocytoma cells

2004 ◽  
Vol 379 (3) ◽  
pp. 641-651 ◽  
Author(s):  
Ian H. BATTY ◽  
Ian N. FLEMING ◽  
C. Peter DOWNES
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Muscarinic Receptors ◽  
Kinase Activity ◽  
Serine Phosphorylation ◽  
Regulatory Subunit ◽  
Insulin Like Growth Factor ◽  
Phosphatase Inhibitor ◽  
Astrocytoma Cells ◽  
1321N1 Astrocytoma

In 1321N1 astrocytoma cells, stimulation of the IGF-1 (insulin-like growth factor-1) receptor increased the association of PI3K [phosphoinositide (PI) 3-kinase] activity with IRS-1 (insulin re-ceptor substrate 1), and increased the cellular concentration of PtdIns(3,4,5)P3. Carbachol, acting on M3 muscarinic receptors, inhibited insulin-, but not PDGF (platelet-derived growth factor)-, stimulated responses by ≈50%. The inhibition of IRS-1-associated PI3K activity by carbachol (i) was rapid (<1 min), persistent (≥60 min) and potent (half-maximal concentration ≈1 µM); (ii) was reproduced by stimuli for several phospholipase-C-coupled receptors; (iii) was prevented by the inhibition of protein kinase C, but not by chelation of intracellular Ca2+; and (iv) was not blocked or reproduced by inhibitors or stimuli respectively of mitogen-activated protein kinase, PI3K, protein kinase B or the mammalian target of rapamycin. However, the effects of carbachol were prevented by sodium vanadate, a protein tyrosine phosphatase inhibitor, and were accompanied by reduced insulin-stimulated IRS-1 tyrosine phosphorylation and recruitment of the 85 kDa regulatory subunit of PI3K to IRS-1, but not by reduced IGF-1 receptor kinase activity. The inhibitory effect of carbachol was reproduced by okadaic acid, a protein serine/threonine phosphatase inhibitor, but not by PDGF, yet all three agents stimulated the serine phosphorylation of IRS-1 at residues Ser312, Ser616 and Ser636/639, albeit to different extents. Thus muscarinic receptors may inhibit insulin signalling by promoting IRS-1 tyrosine dephosphorylation and/or by uncoupling IRS-1 from the stimulated IGF-1 receptor by stimulating IRS-1 serine phosphorylation. However, the proportion of IRS-1 molecules phosphorylated at a particular site or the phosphorylation of additional IRS-1 serine residues other than those noted above must be important.

scholarly journals Genetic manipulation: Rifkin strikes against gene transfer experiments

Nature ◽  
1984 ◽  
Vol 311 (5986) ◽  
pp. 495-495
Author(s):  
Tim Beardsley
Keyword(s):  
Gene Transfer ◽  
Genetic Manipulation
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