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Nicola Neff - One of the best experts on this subject based on the ideXlab platform.
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K 252a promotes survival and choline acetyltransferase activity in striatal and basal forebrain neuronal cultures
Journal of Neurochemistry, 2002Co-Authors: Marcie A Glicksman, M E Forbes, J E Prantner, Nicola NeffAbstract:: The organic molecule K-252a promoted cell survival, neurite outgrowth, and increased choline acetyltransferase (ChAT) activity in rat embryonic striatal and basal forebrain cultures in a concentration-dependent manner. A two- to threefold increase in survival was observed at 75 nM K-252a in both systems. A single application of K-252a at culture initiation prevented substantial (> 60%) cell death that otherwise occurred after 4 days in striatal or basal forebrain cultures. A 5-h exposure of striatal or basal forebrain cells to K-252a, followed by its removal, resulted in survival equivalent to that observed in cultures continually maintained in its presence. This is in contrast to results found with a 5-h exposure of basal forebrain cultures to nerve growth factor (NGF). Acute exposure of basal forebrain cultures to K-252a, but not to NGF, increased ChAT activity, indicating that NGF was required the entire culture period for maximum activity. Striatal cholinergic and GABAergic neurons were among the neurons rescued by K-252a. Of the protein growth factors tested in striatal cultures (ciliary neurotrophic factor, neurotrophin-3, NGF, brain-derived neurotrophic factor, interleuKin-2, basic fibroblast growth factor), only brain-derived neurotrophic factor promoted survival. The enhancement of survival and ChAT activity of basal forebrain and striatal neurons by K-252a defines additional populations of neurons in which survival and/or differentiation is regulated by a K-252a-responsive mechanism. The above results expand the potential therapeutic targets for these molecules for the treatment of neuro-degenerative diseases.
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K‐252a Promotes Survival and Choline Acetyltransferase Activity in Striatal and Basal Forebrain Neuronal Cultures
Journal of Neurochemistry, 2002Co-Authors: Marcie A Glicksman, M E Forbes, J E Prantner, Nicola NeffAbstract:: The organic molecule K-252a promoted cell survival, neurite outgrowth, and increased choline acetyltransferase (ChAT) activity in rat embryonic striatal and basal forebrain cultures in a concentration-dependent manner. A two- to threefold increase in survival was observed at 75 nM K-252a in both systems. A single application of K-252a at culture initiation prevented substantial (> 60%) cell death that otherwise occurred after 4 days in striatal or basal forebrain cultures. A 5-h exposure of striatal or basal forebrain cells to K-252a, followed by its removal, resulted in survival equivalent to that observed in cultures continually maintained in its presence. This is in contrast to results found with a 5-h exposure of basal forebrain cultures to nerve growth factor (NGF). Acute exposure of basal forebrain cultures to K-252a, but not to NGF, increased ChAT activity, indicating that NGF was required the entire culture period for maximum activity. Striatal cholinergic and GABAergic neurons were among the neurons rescued by K-252a. Of the protein growth factors tested in striatal cultures (ciliary neurotrophic factor, neurotrophin-3, NGF, brain-derived neurotrophic factor, interleuKin-2, basic fibroblast growth factor), only brain-derived neurotrophic factor promoted survival. The enhancement of survival and ChAT activity of basal forebrain and striatal neurons by K-252a defines additional populations of neurons in which survival and/or differentiation is regulated by a K-252a-responsive mechanism. The above results expand the potential therapeutic targets for these molecules for the treatment of neuro-degenerative diseases.
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K 252a induces tyrosine phosphorylation of the focal adhesion Kinase and neurite outgrowth in human neuroblastoma sh sy5y cells
Journal of Neurochemistry, 2002Co-Authors: Anna C Maroney, Nicola Neff, Marcie A Glicksman, Lorraine Lipfert, Elizabeth M Forbes, Robert Siman, Craig A DionneAbstract:: The protein Kinase inhibitor K-252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chicK dorsal root ganglion cultures. To determine the mechanism by which K-252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH-SY5Y. K-252a induced neurite outgrowth in a dose-dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125- and 140-KDa proteins. The phosphorylation events were independent of protein Kinase C inhibition because down-regulation of protein Kinase C by long-term treatment with phorbol ester did not prevent K-252a-induced tyrosine phosphorylation. Similarly, the protein Kinase C inhibitors H7, GF-109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine Kinase (FaK). Induction of phosphorylation coincided with increased FaK activity and appeared to be independent of ligand/integrin interaction. The induction of FaK phosphorylation by K-252a was also observed in LA-N-5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein Kinase C-independent induction of tyrosine phosphorylation and the identification of FaK as a substrate of K-252a-induced tyrosine Kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.
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K‐252a Induces Tyrosine Phosphorylation of the Focal Adhesion Kinase and Neurite Outgrowth in Human Neuroblastoma SH‐SY5Y Cells
Journal of Neurochemistry, 2002Co-Authors: Anna C Maroney, Nicola Neff, Marcie A Glicksman, Lorraine Lipfert, Robert Siman, M. Elizabeth Forbes, Craig A DionneAbstract:: The protein Kinase inhibitor K-252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chicK dorsal root ganglion cultures. To determine the mechanism by which K-252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH-SY5Y. K-252a induced neurite outgrowth in a dose-dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125- and 140-KDa proteins. The phosphorylation events were independent of protein Kinase C inhibition because down-regulation of protein Kinase C by long-term treatment with phorbol ester did not prevent K-252a-induced tyrosine phosphorylation. Similarly, the protein Kinase C inhibitors H7, GF-109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine Kinase (FaK). Induction of phosphorylation coincided with increased FaK activity and appeared to be independent of ligand/integrin interaction. The induction of FaK phosphorylation by K-252a was also observed in LA-N-5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein Kinase C-independent induction of tyrosine phosphorylation and the identification of FaK as a substrate of K-252a-induced tyrosine Kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.
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neurotrophic 3 9 bis alKylthio methyl and bis alKoxymethyl K 252a derivatives
Journal of Medicinal Chemistry, 1997Co-Authors: Masami Kaneko, Yuzuru Matsuda, Yutaka Saito, Hiromitsu Saito, Tadashi Matsumoto, Jeffry L Vaught, Craig A Dionne, Thelma S Angeles, Marcie A Glicksman, Nicola NeffAbstract:A series of 3,9-disubstituted [(alKylthio)methyl]- and (alKoxymethyl)-K-252a derivatives was synthesized with the aim of enhancing and separating the neurotrophic properties from the undesirable NGF (trK A Kinase) and PKC inhibitory activities of K-252a. Data from this series reveal that substitution in the 3- and 9-positions of K-252a with these groups reduces trK A Kinase inhibitory properties approximately 100- to >500-fold while maintaining or in certain cases enhancing the neurotrophic activity. From this research, 3,9-bis[(ethylthio)methyl]-K-252a (8) was identified as a potent and selective neurotrophic agent in vitro as measured by enhancement of choline acetyltransferase activity in embryonic rat spinal cord and basal forebrain cultures. Compound 8 was found to have weaK Kinase inhibitory activity for trK A, protein Kinase C, protein Kinase A, and myosin light chain Kinase. On the basis of the in vitro profile, 8 was evaluated in in vivo models suggestive of neurological diseases. Compound 8 was ...
Craig A Dionne - One of the best experts on this subject based on the ideXlab platform.
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K‐252a Induces Tyrosine Phosphorylation of the Focal Adhesion Kinase and Neurite Outgrowth in Human Neuroblastoma SH‐SY5Y Cells
Journal of Neurochemistry, 2002Co-Authors: Anna C Maroney, Nicola Neff, Marcie A Glicksman, Lorraine Lipfert, Robert Siman, M. Elizabeth Forbes, Craig A DionneAbstract:: The protein Kinase inhibitor K-252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chicK dorsal root ganglion cultures. To determine the mechanism by which K-252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH-SY5Y. K-252a induced neurite outgrowth in a dose-dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125- and 140-KDa proteins. The phosphorylation events were independent of protein Kinase C inhibition because down-regulation of protein Kinase C by long-term treatment with phorbol ester did not prevent K-252a-induced tyrosine phosphorylation. Similarly, the protein Kinase C inhibitors H7, GF-109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine Kinase (FaK). Induction of phosphorylation coincided with increased FaK activity and appeared to be independent of ligand/integrin interaction. The induction of FaK phosphorylation by K-252a was also observed in LA-N-5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein Kinase C-independent induction of tyrosine phosphorylation and the identification of FaK as a substrate of K-252a-induced tyrosine Kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.
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K 252a induces tyrosine phosphorylation of the focal adhesion Kinase and neurite outgrowth in human neuroblastoma sh sy5y cells
Journal of Neurochemistry, 2002Co-Authors: Anna C Maroney, Nicola Neff, Marcie A Glicksman, Lorraine Lipfert, Elizabeth M Forbes, Robert Siman, Craig A DionneAbstract:: The protein Kinase inhibitor K-252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chicK dorsal root ganglion cultures. To determine the mechanism by which K-252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH-SY5Y. K-252a induced neurite outgrowth in a dose-dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125- and 140-KDa proteins. The phosphorylation events were independent of protein Kinase C inhibition because down-regulation of protein Kinase C by long-term treatment with phorbol ester did not prevent K-252a-induced tyrosine phosphorylation. Similarly, the protein Kinase C inhibitors H7, GF-109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine Kinase (FaK). Induction of phosphorylation coincided with increased FaK activity and appeared to be independent of ligand/integrin interaction. The induction of FaK phosphorylation by K-252a was also observed in LA-N-5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein Kinase C-independent induction of tyrosine phosphorylation and the identification of FaK as a substrate of K-252a-induced tyrosine Kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.
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Kinetics of trKa tyrosine Kinase activity and inhibition by K 252a
Archives of Biochemistry and Biophysics, 1998Co-Authors: Thelma S Angeles, Shi X Yang, Catherine Steffler, Craig A DionneAbstract:Abstract The Kinetic mechanism of the trK receptor-linKed tyrosine Kinase was determined using a baculovirus expressed trK Kinase domain and a bacterially expressed phospholipase C-γ/glutathione S -transferase (PLC-γ/GST) fusion protein as substrate. Product and dead-end inhibition studies indicate an ordered association of substrates to trKA Kinase with the nucleotide ATP binding prior to the exogenous substrate PLC-γ/GST, followed by release of the phosphorylated PLC-γ/GST product prior to release of ADP (sequential ordered bi–bi mechanism). This is in contrast to the reported Kinetic mechanisms of closely related EGF receptor and insulin receptor Kinases which appear to proceed via a rapid equilibrium random mechanism. The indolocarbazole K-252a, which was previously shown to be a potent and relatively selective inhibitor of trK Kinase activity, acts as a competitive inhibitor with respect to ATP. The data suggest that potent and selective Kinase inhibitors can be rationally designed by exploring subtle variations surrounding the nucleotide binding sites of receptor tyrosine Kinases.
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neurotrophic 3 9 bis alKylthio methyl and bis alKoxymethyl K 252a derivatives
Journal of Medicinal Chemistry, 1997Co-Authors: Masami Kaneko, Yuzuru Matsuda, Yutaka Saito, Hiromitsu Saito, Tadashi Matsumoto, Jeffry L Vaught, Craig A Dionne, Thelma S Angeles, Marcie A Glicksman, Nicola NeffAbstract:A series of 3,9-disubstituted [(alKylthio)methyl]- and (alKoxymethyl)-K-252a derivatives was synthesized with the aim of enhancing and separating the neurotrophic properties from the undesirable NGF (trK A Kinase) and PKC inhibitory activities of K-252a. Data from this series reveal that substitution in the 3- and 9-positions of K-252a with these groups reduces trK A Kinase inhibitory properties approximately 100- to >500-fold while maintaining or in certain cases enhancing the neurotrophic activity. From this research, 3,9-bis[(ethylthio)methyl]-K-252a (8) was identified as a potent and selective neurotrophic agent in vitro as measured by enhancement of choline acetyltransferase activity in embryonic rat spinal cord and basal forebrain cultures. Compound 8 was found to have weaK Kinase inhibitory activity for trK A, protein Kinase C, protein Kinase A, and myosin light chain Kinase. On the basis of the in vitro profile, 8 was evaluated in in vivo models suggestive of neurological diseases. Compound 8 was ...
Kohzaburo Fujikawayamamoto - One of the best experts on this subject based on the ideXlab platform.
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polyploidization of mouse h 1 es cells by demecolcine and K 252a
Cytologia, 2006Co-Authors: Kohzaburo Fujikawayamamoto, Minoru Miyagoshi, Hiroko YamagishiAbstract:Mouse H-1 (ES) cells (H-1 cells) were examined for polyploidization by demecolcine and K-252a. H-1 cells were highly polyploidized by both demecolcine and K-252a, and died 3 d after their addition, suggesting that polyploidized H-1 cells are sensitive to these drugs. The polyploidized H-1 cells showed alKaline phosphatase activity, suggesting that the pluripotent potential of H-1 cells was maintained through out polyploidization. Although the polyploidized H-1 cells became tentatively diploid or tetraploid after the drug removal, the tetraploid H-1 cells reverted to diploid cells within a month.
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establishment of a triploid v79 cell line from tetraploid cells obtained through polyploidization using K 252a
Cell Proliferation, 2002Co-Authors: Kohzaburo Fujikawayamamoto, Hiroko Yamagishi, Minoru MiyagoshiAbstract:Triploid V79 cells were established from tetraploid cells. Diploid V79 cells were polyploidized by K-252a, an inhibitor of protein Kinases, and then released from the drug for 10 days. At that time, the cell population was a mixture of diploid and tetraploid cells. Triploid cells were obtained through the cloning of tetraploid cells. They had 33 chromosomes (1.5 times the diploid number) and showed a Karyotype of three homologueous chromosomes. The duration of the G 1 , S and G 2 /M phases was almost the same as for diploid cells. The cell volume of triploid V79 cells was about two times that of the diploid cells. An explanation for the diploid-tetraploid-triploid transition is proposed.
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establishment of a tetraploid meth a cell line through polyploidization by demecolcine but not by staurosporine K 252a and paclitaxel
Cell Proliferation, 2001Co-Authors: Kohzaburo Fujikawayamamoto, Hiroko Yamagishi, Chie Ohdoi, S Wang, H Murano, Teruaki IkedaAbstract:: Polyploid cells are made by DNA reduplication without cell division, however, it is not easy to establish polyploid mammalian cell lines. It is worth studying the difference in cell character between hyperploid and parent cell lines. Meth-A cells were polyploidized by demecolcine, K-252a, staurosporine and paclitaxel. The cell-cycle responses of highly polyploid Meth-A cells after the removal of the drugs were examined by flow cytometry (FCM). Meth-A cells were highly polyploidized by these drugs. The polyploid Meth-A cells gradually decreased in ploidy after the drug release. A tetraploid Meth-A cell line was established only from the demecolcine-induced polyploid Meth-A cells. The duration of G1, S and G2/M phases of the tetraploid cell line were mostly the same as those of the parent diploid cells, except that the G2/M phase was 1.5 h longer. The chromosome number of tetraploid Meth-A cell line was about twice of the diploidy. A tetraploid Meth-A cell line was established.
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different responses of polyploidized v79 cells after removal of two drugs demecolcine and K 252a
Cell Structure and Function, 2000Co-Authors: Kohzaburo Fujikawayamamoto, Hiroko Yamagishi, Zhi-ping Zong, Chie Ohdoi, Shiyong WangAbstract:To examine whether or not cells polyploidized by different mechanisms behave in a different manner after drug removal, V79 Chinese hamster cells were assessed by flow cytometry (FCM) after their polyploidization by demecolcine and K-252a, inhibitors of spindle-fiber formation and protein Kinase, respectively. Cell cycle analysis of DNA histograms of V79 cells before and after the drug release was performed. With both drugs, the ploidy of V79 cells increased just after the drug removal and was maintained for a weeK. A difference was evident 10 days after the release. Tetraploid cells were the main population from 10 to 18 days after the release of K-252a, but not demecolcine. Cell cycle parameters were almost the same in pseudo diploid and tetraploid V79 cells, except for the tetraploid S phase which was 2h longer.
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hyperploidization of v79 cells by K 252a in comparison with demecolcine
Cell Structure and Function, 1993Co-Authors: Kohzaburo Fujikawayamamoto, Kouhei Teraoka, Shizuo OdashimaAbstract:: The cellular responses of the V79 cell cycle to K-252a, a protein Kinase inhibitor, were examined by flow cytometry (FCM) and by time-lapse videomicrography. At a concentration of 0.5 microM of K-252a for 72 h, V79 cells became hyperploid, having above 32c DNA content. The cycle times in the hyperploidizing process were 11.2 (4c-8c), 16.3 (8c-16c) and 19.3 (16c-32c) hours, values which were less than twice that of the control (10.5 h). After the washout of K-252a, the DNA content was widely distributed in the V79 cell population and hyperploid V79 cells reduced DNA content through a variety of cell division modes. These cellular responses to hyperploidization of V79 cells by K-252a were in reasonable agreement with those by demecolcine.
Marcie A Glicksman - One of the best experts on this subject based on the ideXlab platform.
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K 252a promotes survival and choline acetyltransferase activity in striatal and basal forebrain neuronal cultures
Journal of Neurochemistry, 2002Co-Authors: Marcie A Glicksman, M E Forbes, J E Prantner, Nicola NeffAbstract:: The organic molecule K-252a promoted cell survival, neurite outgrowth, and increased choline acetyltransferase (ChAT) activity in rat embryonic striatal and basal forebrain cultures in a concentration-dependent manner. A two- to threefold increase in survival was observed at 75 nM K-252a in both systems. A single application of K-252a at culture initiation prevented substantial (> 60%) cell death that otherwise occurred after 4 days in striatal or basal forebrain cultures. A 5-h exposure of striatal or basal forebrain cells to K-252a, followed by its removal, resulted in survival equivalent to that observed in cultures continually maintained in its presence. This is in contrast to results found with a 5-h exposure of basal forebrain cultures to nerve growth factor (NGF). Acute exposure of basal forebrain cultures to K-252a, but not to NGF, increased ChAT activity, indicating that NGF was required the entire culture period for maximum activity. Striatal cholinergic and GABAergic neurons were among the neurons rescued by K-252a. Of the protein growth factors tested in striatal cultures (ciliary neurotrophic factor, neurotrophin-3, NGF, brain-derived neurotrophic factor, interleuKin-2, basic fibroblast growth factor), only brain-derived neurotrophic factor promoted survival. The enhancement of survival and ChAT activity of basal forebrain and striatal neurons by K-252a defines additional populations of neurons in which survival and/or differentiation is regulated by a K-252a-responsive mechanism. The above results expand the potential therapeutic targets for these molecules for the treatment of neuro-degenerative diseases.
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K‐252a Promotes Survival and Choline Acetyltransferase Activity in Striatal and Basal Forebrain Neuronal Cultures
Journal of Neurochemistry, 2002Co-Authors: Marcie A Glicksman, M E Forbes, J E Prantner, Nicola NeffAbstract:: The organic molecule K-252a promoted cell survival, neurite outgrowth, and increased choline acetyltransferase (ChAT) activity in rat embryonic striatal and basal forebrain cultures in a concentration-dependent manner. A two- to threefold increase in survival was observed at 75 nM K-252a in both systems. A single application of K-252a at culture initiation prevented substantial (> 60%) cell death that otherwise occurred after 4 days in striatal or basal forebrain cultures. A 5-h exposure of striatal or basal forebrain cells to K-252a, followed by its removal, resulted in survival equivalent to that observed in cultures continually maintained in its presence. This is in contrast to results found with a 5-h exposure of basal forebrain cultures to nerve growth factor (NGF). Acute exposure of basal forebrain cultures to K-252a, but not to NGF, increased ChAT activity, indicating that NGF was required the entire culture period for maximum activity. Striatal cholinergic and GABAergic neurons were among the neurons rescued by K-252a. Of the protein growth factors tested in striatal cultures (ciliary neurotrophic factor, neurotrophin-3, NGF, brain-derived neurotrophic factor, interleuKin-2, basic fibroblast growth factor), only brain-derived neurotrophic factor promoted survival. The enhancement of survival and ChAT activity of basal forebrain and striatal neurons by K-252a defines additional populations of neurons in which survival and/or differentiation is regulated by a K-252a-responsive mechanism. The above results expand the potential therapeutic targets for these molecules for the treatment of neuro-degenerative diseases.
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K 252a induces tyrosine phosphorylation of the focal adhesion Kinase and neurite outgrowth in human neuroblastoma sh sy5y cells
Journal of Neurochemistry, 2002Co-Authors: Anna C Maroney, Nicola Neff, Marcie A Glicksman, Lorraine Lipfert, Elizabeth M Forbes, Robert Siman, Craig A DionneAbstract:: The protein Kinase inhibitor K-252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chicK dorsal root ganglion cultures. To determine the mechanism by which K-252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH-SY5Y. K-252a induced neurite outgrowth in a dose-dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125- and 140-KDa proteins. The phosphorylation events were independent of protein Kinase C inhibition because down-regulation of protein Kinase C by long-term treatment with phorbol ester did not prevent K-252a-induced tyrosine phosphorylation. Similarly, the protein Kinase C inhibitors H7, GF-109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine Kinase (FaK). Induction of phosphorylation coincided with increased FaK activity and appeared to be independent of ligand/integrin interaction. The induction of FaK phosphorylation by K-252a was also observed in LA-N-5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein Kinase C-independent induction of tyrosine phosphorylation and the identification of FaK as a substrate of K-252a-induced tyrosine Kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.
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K‐252a Induces Tyrosine Phosphorylation of the Focal Adhesion Kinase and Neurite Outgrowth in Human Neuroblastoma SH‐SY5Y Cells
Journal of Neurochemistry, 2002Co-Authors: Anna C Maroney, Nicola Neff, Marcie A Glicksman, Lorraine Lipfert, Robert Siman, M. Elizabeth Forbes, Craig A DionneAbstract:: The protein Kinase inhibitor K-252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chicK dorsal root ganglion cultures. To determine the mechanism by which K-252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH-SY5Y. K-252a induced neurite outgrowth in a dose-dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125- and 140-KDa proteins. The phosphorylation events were independent of protein Kinase C inhibition because down-regulation of protein Kinase C by long-term treatment with phorbol ester did not prevent K-252a-induced tyrosine phosphorylation. Similarly, the protein Kinase C inhibitors H7, GF-109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine Kinase (FaK). Induction of phosphorylation coincided with increased FaK activity and appeared to be independent of ligand/integrin interaction. The induction of FaK phosphorylation by K-252a was also observed in LA-N-5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein Kinase C-independent induction of tyrosine phosphorylation and the identification of FaK as a substrate of K-252a-induced tyrosine Kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.
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neurotrophic 3 9 bis alKylthio methyl and bis alKoxymethyl K 252a derivatives
Journal of Medicinal Chemistry, 1997Co-Authors: Masami Kaneko, Yuzuru Matsuda, Yutaka Saito, Hiromitsu Saito, Tadashi Matsumoto, Jeffry L Vaught, Craig A Dionne, Thelma S Angeles, Marcie A Glicksman, Nicola NeffAbstract:A series of 3,9-disubstituted [(alKylthio)methyl]- and (alKoxymethyl)-K-252a derivatives was synthesized with the aim of enhancing and separating the neurotrophic properties from the undesirable NGF (trK A Kinase) and PKC inhibitory activities of K-252a. Data from this series reveal that substitution in the 3- and 9-positions of K-252a with these groups reduces trK A Kinase inhibitory properties approximately 100- to >500-fold while maintaining or in certain cases enhancing the neurotrophic activity. From this research, 3,9-bis[(ethylthio)methyl]-K-252a (8) was identified as a potent and selective neurotrophic agent in vitro as measured by enhancement of choline acetyltransferase activity in embryonic rat spinal cord and basal forebrain cultures. Compound 8 was found to have weaK Kinase inhibitory activity for trK A, protein Kinase C, protein Kinase A, and myosin light chain Kinase. On the basis of the in vitro profile, 8 was evaluated in in vivo models suggestive of neurological diseases. Compound 8 was ...
Minoru Miyagoshi - One of the best experts on this subject based on the ideXlab platform.
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Polyploidization of 2nH1 (ES) cells by K-252a and staurosporine
Human Cell, 2007Co-Authors: Kohzaburo Fujikawa-yamamoto, Minoru Miyagoshi, Hiroko YamagishiAbstract:Mouse 2nH1 (ES) cells were examined for polyploidization using K-252a and staurosporine. Though 2nH1 cells were polyploidized by both K-252a and staurosporine, tetraploid cells, 4nH1K cells, were obtained only from cell populations exposed to K-252a. The probability of successful establishment of tetraploid cells was 2/9, suggesting that the highly polyploidized—tetraploid transition might occur infrequently. Cell cycle parameters of 4nH1 K cells were almost the same as those of 2nH1 cells, suggesting that the rate of DNA synthesis was about twice that of the diploid cells. The cell volume of 4nH1K cells was about twice of that of diploid cells, indicating that 4nH1K cells contained about twice as much total intracellular material as 2nH1 cells. The morphology of the 4nH1 K cells was flagstone-liKe, thus differing from that of the spindle-shaped 2nH1 cells, suggesting that morphological transformation occurred during the diploid-tetraploid transition. 4nH1K cells exhibited alKaline phosphatase activity and formed teratocarcinomas, implying that they were pluripotent. These characteristics of 4nH1K cells were similar to those of tetraploid 4nH1 cells that have been established through polyploidization by demecolcine, suggesting that 4nH1K and 4nH1 cells are similar irrespective of the different mechanisms of polyploidization.
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polyploidization of mouse h 1 es cells by demecolcine and K 252a
Cytologia, 2006Co-Authors: Kohzaburo Fujikawayamamoto, Minoru Miyagoshi, Hiroko YamagishiAbstract:Mouse H-1 (ES) cells (H-1 cells) were examined for polyploidization by demecolcine and K-252a. H-1 cells were highly polyploidized by both demecolcine and K-252a, and died 3 d after their addition, suggesting that polyploidized H-1 cells are sensitive to these drugs. The polyploidized H-1 cells showed alKaline phosphatase activity, suggesting that the pluripotent potential of H-1 cells was maintained through out polyploidization. Although the polyploidized H-1 cells became tentatively diploid or tetraploid after the drug removal, the tetraploid H-1 cells reverted to diploid cells within a month.
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establishment of a triploid v79 cell line from tetraploid cells obtained through polyploidization using K 252a
Cell Proliferation, 2002Co-Authors: Kohzaburo Fujikawayamamoto, Hiroko Yamagishi, Minoru MiyagoshiAbstract:Triploid V79 cells were established from tetraploid cells. Diploid V79 cells were polyploidized by K-252a, an inhibitor of protein Kinases, and then released from the drug for 10 days. At that time, the cell population was a mixture of diploid and tetraploid cells. Triploid cells were obtained through the cloning of tetraploid cells. They had 33 chromosomes (1.5 times the diploid number) and showed a Karyotype of three homologueous chromosomes. The duration of the G 1 , S and G 2 /M phases was almost the same as for diploid cells. The cell volume of triploid V79 cells was about two times that of the diploid cells. An explanation for the diploid-tetraploid-triploid transition is proposed.
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Establishment of a triploid V79 cell line from tetraploid cells obtained through polyploidization using K‐252a
Cell Proliferation, 2002Co-Authors: Kohzaburo Fujikawa-yamamoto, Hiroko Yamagishi, Minoru MiyagoshiAbstract:Triploid V79 cells were established from tetraploid cells. Diploid V79 cells were polyploidized by K-252a, an inhibitor of protein Kinases, and then released from the drug for 10 days. At that time, the cell population was a mixture of diploid and tetraploid cells. Triploid cells were obtained through the cloning of tetraploid cells. They had 33 chromosomes (1.5 times the diploid number) and showed a Karyotype of three homologueous chromosomes. The duration of the G 1 , S and G 2 /M phases was almost the same as for diploid cells. The cell volume of triploid V79 cells was about two times that of the diploid cells. An explanation for the diploid-tetraploid-triploid transition is proposed.