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Imola Wilhelm Péter Nagyőszi Attila E. Farkas Pierre‐Olivier Couraud Ignacio A. Romero Babette Weksler Csilla Fazakas Ngo Thi Khue Dung Sándor Bottka Hannelore Bauer Hans‐Christian Bauer István A. Krizbai 《Journal of neurochemistry》2008,107(1):116-126
Because of the relative impermeability of the blood‐brain barrier (BBB), many drugs are unable to reach the CNS in therapeutically relevant concentration. One method to deliver drugs to the CNS is the osmotic opening of the BBB using mannitol. Hyperosmotic mannitol induces a strong phosphorylation on tyrosine residues in a broad spectrum of proteins in cerebral endothelial cells, the principal components of the BBB. Previously, we have shown that among targets of tyrosine phosphorylation are β‐catenin, extracellular signal‐regulated kinase 1/2 and the non‐receptor tyrosine kinase Src. The aim of this study was to identify new signalling pathways activated by hypertonicity in cerebral endothelial cells. Using an antibody array and immunoprecipitation we identified the receptor tyrosine kinase Axl to become tyrosine phosphorylated in response to hyperosmotic mannitol. Besides activation, Axl was also cleaved in response to osmotic stress. Degradation of Axl proved to be metalloproteinase‐ and proteasome‐dependent and resulted in 50–55 kDa C‐terminal products which remained phosphorylated even after degradation. Specific knockdown of Axl increased the rate of apoptosis in hyperosmotic mannitol‐treated cells; therefore, we assume that activation of Axl may be a protective mechanism against hypertonicity‐induced apoptosis. Our results identify Axl as an important element of osmotic stress‐induced signalling. 相似文献
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Lymphoid cells expressing sufficient levels of Bcl-2 or E1B-19K are known to resist to induction of apoptosis in glutamine-free
or nutrient-limited batch cultures. However, despite the increased viability and prolonged stationary phase achieved in batch
culture, product yields are not necessarily improved. Here we have found that expression of E1B-19K in NS/0 myeloma cells
cultivated in the presence of certain cell cycle modulators could result in a significant increase in MAb productivity as
compared to untransfected control cells. The use of E1B-19K significantly enhanced cell survival in the presence of osmolytes
(sorbitol, NaCl), DNA synthesis inhibitors (hydroxyurea, excess thymidine), and the cell culture additive OptiMAb™. E1B-19K
myelomas cultivated in the presence of NaCl or OptiMAb™ accumulated in the G1 phase, while those arrested with excess thymidine
were blocked in all phases. Interestingly, control NS/0 cells treated with these agents were found to die in a cell-cycle
specific manner. Thus, while all G1 and most S phase cells quickly underwent apoptosis, G2/M cells remained alive and maintained
MAb secretion for more than 10 days if supplied with adequate nutrients. For both control and E1B-19K cells, incubation with
sorbitol or hydroxyurea was detrimental for MAb secretion, while addition of NaCl, excess thymidine and OptiMAb™ resulted
in an increased specific MAb productivity as compared to the batch culture. However, this increase resulted in an improvement
of final MAb yields only in the case of OptiMAb™. The extension of viability conferred by E1B-19K allowed to further improve
the final MAb yield obtained using OptiMAb™ with a 3.3-fold increase for E1B-19K cells as compared to 1.8-fold for control
NS/0 cells.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
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