The Experts below are selected from a list of 1113 Experts worldwide ranked by ideXlab platform
Masafumi Yamaguchi - One of the best experts on this subject based on the ideXlab platform.
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knockdown of shwachman diamond syndrome gene sbds induces increased expression of galectin 1 and impaired cell growth
Blood, 2012Co-Authors: Masafumi Yamaguchi, Kingo Fujimura, Hanae Togayamaguchi, Hirokazu Kanegane, Naoki OkamuraAbstract:Abstract 2360 Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11. We have previously reported the Shwachman-Bodian-Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells which established by SBDS shRNAi were sensitive to apoptotic stimuli, and led to growth inhibition, indicating that SBDS acts to maintain survival of Granulocyte Precursor cells (Exp Hematol 35;579,2007). The precise mechanism by which the loss of SBDS inhibits growth of cells remains elusive. In order to clarify the impaired cell growth of SBDS knockdown cells, we analyzed two SDS patients (c.183_184TA>CT and c.258+2T>C) derived EB virus transformed lymphoblast cells (LCL). The growth of both LCL-SDS cell lines was considerably lower than control donor cells (LCL-C) which occurred in within 3 days of culture (1.4×10 6 cells/ml in LCL-C vs 5×10 5 cells/lm in LCL-SDS). LCL-C cells divided until 5 days, however, growth of LCL-SDS cells was saturated in 3days. When LCL-SDS cells were seeded to the fresh medium, LCL-SDS cells proliferated again. Conditional medium from 5 days SDS-LCL cell culture was then added to the culture of both LCL-C and LCL-SDS cell. This LCL-SDS conditional medium inhibited both LCL-C and LCL-SDS cell growth (50% and 60%, respectively), suggesting that growth inhibitors were secreted from LCL-SDS cells. In order to find growth inhibitors, we performed differential display. By annealing control primer based GeneFishing PCR screening, we found galectin-1 mRNA level was increased in LCL-SDS cells. We also confirmed that Galectin-1(Gal1) protein expression was markedly increased in LCL-SDS cells by western blot and conforcal microscopy. Gal1 was found to membrane bound, and it is plausible that Gal1 was secreted to the medium. In order to isolate Gal1 protein from medium, medium was passed through lactose agarose. Gal1 protein was purified from LCL-SDS cell culture medium, not from LCL-C cells. The inhibitory effect of Gal1 was confirmed using recombinant human Galectin-1 (rhGal1), which had similar dynamics to that of conditional medium from LCL-SDS cells. rhGal1 the proliferation of both LCL-C and LCL-SDS cells in a dose dependent manner. After exposure to rhGal1, Annexin V positive cells were increased in LCL-C cells (13.78±2.09% in control vs 16.83±2.81% in rhGal1, p=0.02). However, there was no difference in Gal1 induced apoptosis between LCL-C and LCL-SDS cells. In order to rescue growth failure of LCL-SDS cells, lactose, which modulates the binding between galectin and its substrate, was supplemented to the medium. Though lactose showed the growth inhibition of LCL cells, the viability of LCL-SDS cells was much higher than LCL-C cells. LCL-SDS cells were easily aggregated, however, the colony of LCL-SDS cell was much smaller in the presence of lactose. We also confirmed that Gal1 protein was overexpressed in SBDS knockdown 32Dcl3 cells, which were established by SBDS shRNAi. Conclusion: Overexpressed Gal1 was found from SDS patient9s derived LCL cells and SBDS shRNAi knockdown 32Dcl3 cells. Gal1 was also found in the conditional medium of LCL-SBDS cells, and secreted Gal1 inhibited the cell proliferation. These results indicated that Galectin-1 partially involved in growth failure of SBDS deficient cells. Disclosures: No relevant conflicts of interest to declare.
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mutations of the sbds gene result in nuclear mislocalization
Blood, 2008Co-Authors: Masafumi Yamaguchi, Kingo Fujimura, Naoki Okamura, Hanae Togayamaguchi, Valentina Svetic, Rajesh ChopraAbstract:Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11. We have previously reported that Shwachman-Bodian- Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells were sensitive to apoptotic stimuli, indicating that SBDS acts to maintain survival of Granulocyte Precursor cells. (Exp Hematol35; 579, 2007). A wide variety of mutations in SBDS gene has been identified, and almost of all patients show truncated immature proteins, p.K62X (c.183_184TA>CT) or p.C84fsX3 (c.258+2T>C). However, it is not yet clear how these truncated proteins affect cellular processes that result in the SDS phenotype. The SBDS protein is localized to the nucleoli but does not have the canonical nuclear localization signal. In order to clarify the molecular basis of pathogenicity of mutated SBDS proteins, we explored the subcellular distribution of normal and mutant SBDS proteins in Hela and 32Dcl3 cells. Using various N-terminal and C-terminal deletion constructs, we found N-terminal region, domain I (1-87 amino acid residue) in particular, was necessary to localize to the nucleus. The disease related mutations (C31W, K33E, N34I, L71P) and the mutations which are conserved among the species in the domain I (E44K, K62E, D70N, E82K) were generated. C31W and N34I mutants failed to localize SBDS to the nuclei. The SV40 derived nuclear localization signal was fused to these mutated SBDS protein, and these proteins were clearly localized to the nuclei. In addition to the mislocalization, the protein expression level of these mutants showed a dramatic decrease compared to the wild type. We also established SBDS wild type and domain I overexpressed 32Dcl3 cell. SBDS wild type overexpressed cells could differentiate to normal neutrophils in the presence of mG-CSF, however domain I overexpressed cells did not differentiate. Almost of all cells showed apoptosis in this domain I overexpressed cells in the presence of mG-CSF, and this was very similar like SBDS RNAi knockdown cells. The localization of endogenous SBDS protein was also analyzed in this domain I overexpressed cells. The domain I was concentrated to nuclei, however endogenous SBDS protein was diffused to cytosol. Conclusions: The present findings enable us to document the nuclear localization signals in SBDS domain I, and that the shuttling protein would promote SBDS to nuclei. These results also showed that mislocalization and/or low expression level of mutated SBDS protein would cause SDS.
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Shwachman-Diamond syndrome is not necessary for the terminal maturation of neutrophils but is important for maintaining viability of Granulocyte Precursors.
Experimental hematology, 2007Co-Authors: Masafumi Yamaguchi, Kingo Fujimura, Naoki Okamura, Hanae Toga, Asim Khwaja, Rajesh ChopraAbstract:Objective Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11, and disease-associated mutations were reported in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SBDS is a member of a highly conserved protein family in diverse species including archaea and eukaryotes. It is widely expressed in many tissues, and its function is still unknown. To investigate the function of the SBDS protein, we undertook loss-of-function experiments in the 32Dcl3 cell line, which has the potential to differentiate to mature neutrophils. Methods SBDS gene was downregulated with lentivirus-based RNAi system. SBDS knockdown cells were analyzed for surface marker expression by flow cytometry and analyzed for the sensitivity to apoptosis-inducing stimuli. Results After culture in Granulocyte colony-stimulating factor (G-CSF)-containing medium for 3 days, 32Dcl3 cells demonstrated normal proliferation but complete downregulation of SBDS protein expression. The SBDS RNAi knockdown cells did not proliferate in G-CSF-containing medium but after 7 days had the appearance of segmented neutrophils. The neutrophil maturation markers were detected on these cells. Undifferentiated SBDS RNAi knockdown cells demonstrated increased apoptosis of undifferentiated cells. Notably, SBDS RNAi knockdown cells demonstrated normal proliferation in interleukin-3-containing medium. Conclusion We have established an SDS model cell line and have used this model to demonstrate that SBDS is not required for neutrophil maturation. However, SBDS knockdown cells were sensitive to apoptotic stimuli, indicating that SBDS acts to maintain survival of Granulocyte Precursor cells.
Rajesh Chopra - One of the best experts on this subject based on the ideXlab platform.
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mutations of the sbds gene result in nuclear mislocalization
Blood, 2008Co-Authors: Masafumi Yamaguchi, Kingo Fujimura, Naoki Okamura, Hanae Togayamaguchi, Valentina Svetic, Rajesh ChopraAbstract:Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11. We have previously reported that Shwachman-Bodian- Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells were sensitive to apoptotic stimuli, indicating that SBDS acts to maintain survival of Granulocyte Precursor cells. (Exp Hematol35; 579, 2007). A wide variety of mutations in SBDS gene has been identified, and almost of all patients show truncated immature proteins, p.K62X (c.183_184TA>CT) or p.C84fsX3 (c.258+2T>C). However, it is not yet clear how these truncated proteins affect cellular processes that result in the SDS phenotype. The SBDS protein is localized to the nucleoli but does not have the canonical nuclear localization signal. In order to clarify the molecular basis of pathogenicity of mutated SBDS proteins, we explored the subcellular distribution of normal and mutant SBDS proteins in Hela and 32Dcl3 cells. Using various N-terminal and C-terminal deletion constructs, we found N-terminal region, domain I (1-87 amino acid residue) in particular, was necessary to localize to the nucleus. The disease related mutations (C31W, K33E, N34I, L71P) and the mutations which are conserved among the species in the domain I (E44K, K62E, D70N, E82K) were generated. C31W and N34I mutants failed to localize SBDS to the nuclei. The SV40 derived nuclear localization signal was fused to these mutated SBDS protein, and these proteins were clearly localized to the nuclei. In addition to the mislocalization, the protein expression level of these mutants showed a dramatic decrease compared to the wild type. We also established SBDS wild type and domain I overexpressed 32Dcl3 cell. SBDS wild type overexpressed cells could differentiate to normal neutrophils in the presence of mG-CSF, however domain I overexpressed cells did not differentiate. Almost of all cells showed apoptosis in this domain I overexpressed cells in the presence of mG-CSF, and this was very similar like SBDS RNAi knockdown cells. The localization of endogenous SBDS protein was also analyzed in this domain I overexpressed cells. The domain I was concentrated to nuclei, however endogenous SBDS protein was diffused to cytosol. Conclusions: The present findings enable us to document the nuclear localization signals in SBDS domain I, and that the shuttling protein would promote SBDS to nuclei. These results also showed that mislocalization and/or low expression level of mutated SBDS protein would cause SDS.
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Shwachman-Diamond syndrome is not necessary for the terminal maturation of neutrophils but is important for maintaining viability of Granulocyte Precursors.
Experimental hematology, 2007Co-Authors: Masafumi Yamaguchi, Kingo Fujimura, Naoki Okamura, Hanae Toga, Asim Khwaja, Rajesh ChopraAbstract:Objective Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11, and disease-associated mutations were reported in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SBDS is a member of a highly conserved protein family in diverse species including archaea and eukaryotes. It is widely expressed in many tissues, and its function is still unknown. To investigate the function of the SBDS protein, we undertook loss-of-function experiments in the 32Dcl3 cell line, which has the potential to differentiate to mature neutrophils. Methods SBDS gene was downregulated with lentivirus-based RNAi system. SBDS knockdown cells were analyzed for surface marker expression by flow cytometry and analyzed for the sensitivity to apoptosis-inducing stimuli. Results After culture in Granulocyte colony-stimulating factor (G-CSF)-containing medium for 3 days, 32Dcl3 cells demonstrated normal proliferation but complete downregulation of SBDS protein expression. The SBDS RNAi knockdown cells did not proliferate in G-CSF-containing medium but after 7 days had the appearance of segmented neutrophils. The neutrophil maturation markers were detected on these cells. Undifferentiated SBDS RNAi knockdown cells demonstrated increased apoptosis of undifferentiated cells. Notably, SBDS RNAi knockdown cells demonstrated normal proliferation in interleukin-3-containing medium. Conclusion We have established an SDS model cell line and have used this model to demonstrate that SBDS is not required for neutrophil maturation. However, SBDS knockdown cells were sensitive to apoptotic stimuli, indicating that SBDS acts to maintain survival of Granulocyte Precursor cells.
Naoki Okamura - One of the best experts on this subject based on the ideXlab platform.
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knockdown of shwachman diamond syndrome gene sbds induces increased expression of galectin 1 and impaired cell growth
Blood, 2012Co-Authors: Masafumi Yamaguchi, Kingo Fujimura, Hanae Togayamaguchi, Hirokazu Kanegane, Naoki OkamuraAbstract:Abstract 2360 Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11. We have previously reported the Shwachman-Bodian-Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells which established by SBDS shRNAi were sensitive to apoptotic stimuli, and led to growth inhibition, indicating that SBDS acts to maintain survival of Granulocyte Precursor cells (Exp Hematol 35;579,2007). The precise mechanism by which the loss of SBDS inhibits growth of cells remains elusive. In order to clarify the impaired cell growth of SBDS knockdown cells, we analyzed two SDS patients (c.183_184TA>CT and c.258+2T>C) derived EB virus transformed lymphoblast cells (LCL). The growth of both LCL-SDS cell lines was considerably lower than control donor cells (LCL-C) which occurred in within 3 days of culture (1.4×10 6 cells/ml in LCL-C vs 5×10 5 cells/lm in LCL-SDS). LCL-C cells divided until 5 days, however, growth of LCL-SDS cells was saturated in 3days. When LCL-SDS cells were seeded to the fresh medium, LCL-SDS cells proliferated again. Conditional medium from 5 days SDS-LCL cell culture was then added to the culture of both LCL-C and LCL-SDS cell. This LCL-SDS conditional medium inhibited both LCL-C and LCL-SDS cell growth (50% and 60%, respectively), suggesting that growth inhibitors were secreted from LCL-SDS cells. In order to find growth inhibitors, we performed differential display. By annealing control primer based GeneFishing PCR screening, we found galectin-1 mRNA level was increased in LCL-SDS cells. We also confirmed that Galectin-1(Gal1) protein expression was markedly increased in LCL-SDS cells by western blot and conforcal microscopy. Gal1 was found to membrane bound, and it is plausible that Gal1 was secreted to the medium. In order to isolate Gal1 protein from medium, medium was passed through lactose agarose. Gal1 protein was purified from LCL-SDS cell culture medium, not from LCL-C cells. The inhibitory effect of Gal1 was confirmed using recombinant human Galectin-1 (rhGal1), which had similar dynamics to that of conditional medium from LCL-SDS cells. rhGal1 the proliferation of both LCL-C and LCL-SDS cells in a dose dependent manner. After exposure to rhGal1, Annexin V positive cells were increased in LCL-C cells (13.78±2.09% in control vs 16.83±2.81% in rhGal1, p=0.02). However, there was no difference in Gal1 induced apoptosis between LCL-C and LCL-SDS cells. In order to rescue growth failure of LCL-SDS cells, lactose, which modulates the binding between galectin and its substrate, was supplemented to the medium. Though lactose showed the growth inhibition of LCL cells, the viability of LCL-SDS cells was much higher than LCL-C cells. LCL-SDS cells were easily aggregated, however, the colony of LCL-SDS cell was much smaller in the presence of lactose. We also confirmed that Gal1 protein was overexpressed in SBDS knockdown 32Dcl3 cells, which were established by SBDS shRNAi. Conclusion: Overexpressed Gal1 was found from SDS patient9s derived LCL cells and SBDS shRNAi knockdown 32Dcl3 cells. Gal1 was also found in the conditional medium of LCL-SBDS cells, and secreted Gal1 inhibited the cell proliferation. These results indicated that Galectin-1 partially involved in growth failure of SBDS deficient cells. Disclosures: No relevant conflicts of interest to declare.
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mutations of the sbds gene result in nuclear mislocalization
Blood, 2008Co-Authors: Masafumi Yamaguchi, Kingo Fujimura, Naoki Okamura, Hanae Togayamaguchi, Valentina Svetic, Rajesh ChopraAbstract:Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11. We have previously reported that Shwachman-Bodian- Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells were sensitive to apoptotic stimuli, indicating that SBDS acts to maintain survival of Granulocyte Precursor cells. (Exp Hematol35; 579, 2007). A wide variety of mutations in SBDS gene has been identified, and almost of all patients show truncated immature proteins, p.K62X (c.183_184TA>CT) or p.C84fsX3 (c.258+2T>C). However, it is not yet clear how these truncated proteins affect cellular processes that result in the SDS phenotype. The SBDS protein is localized to the nucleoli but does not have the canonical nuclear localization signal. In order to clarify the molecular basis of pathogenicity of mutated SBDS proteins, we explored the subcellular distribution of normal and mutant SBDS proteins in Hela and 32Dcl3 cells. Using various N-terminal and C-terminal deletion constructs, we found N-terminal region, domain I (1-87 amino acid residue) in particular, was necessary to localize to the nucleus. The disease related mutations (C31W, K33E, N34I, L71P) and the mutations which are conserved among the species in the domain I (E44K, K62E, D70N, E82K) were generated. C31W and N34I mutants failed to localize SBDS to the nuclei. The SV40 derived nuclear localization signal was fused to these mutated SBDS protein, and these proteins were clearly localized to the nuclei. In addition to the mislocalization, the protein expression level of these mutants showed a dramatic decrease compared to the wild type. We also established SBDS wild type and domain I overexpressed 32Dcl3 cell. SBDS wild type overexpressed cells could differentiate to normal neutrophils in the presence of mG-CSF, however domain I overexpressed cells did not differentiate. Almost of all cells showed apoptosis in this domain I overexpressed cells in the presence of mG-CSF, and this was very similar like SBDS RNAi knockdown cells. The localization of endogenous SBDS protein was also analyzed in this domain I overexpressed cells. The domain I was concentrated to nuclei, however endogenous SBDS protein was diffused to cytosol. Conclusions: The present findings enable us to document the nuclear localization signals in SBDS domain I, and that the shuttling protein would promote SBDS to nuclei. These results also showed that mislocalization and/or low expression level of mutated SBDS protein would cause SDS.
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Shwachman-Diamond syndrome is not necessary for the terminal maturation of neutrophils but is important for maintaining viability of Granulocyte Precursors.
Experimental hematology, 2007Co-Authors: Masafumi Yamaguchi, Kingo Fujimura, Naoki Okamura, Hanae Toga, Asim Khwaja, Rajesh ChopraAbstract:Objective Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11, and disease-associated mutations were reported in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SBDS is a member of a highly conserved protein family in diverse species including archaea and eukaryotes. It is widely expressed in many tissues, and its function is still unknown. To investigate the function of the SBDS protein, we undertook loss-of-function experiments in the 32Dcl3 cell line, which has the potential to differentiate to mature neutrophils. Methods SBDS gene was downregulated with lentivirus-based RNAi system. SBDS knockdown cells were analyzed for surface marker expression by flow cytometry and analyzed for the sensitivity to apoptosis-inducing stimuli. Results After culture in Granulocyte colony-stimulating factor (G-CSF)-containing medium for 3 days, 32Dcl3 cells demonstrated normal proliferation but complete downregulation of SBDS protein expression. The SBDS RNAi knockdown cells did not proliferate in G-CSF-containing medium but after 7 days had the appearance of segmented neutrophils. The neutrophil maturation markers were detected on these cells. Undifferentiated SBDS RNAi knockdown cells demonstrated increased apoptosis of undifferentiated cells. Notably, SBDS RNAi knockdown cells demonstrated normal proliferation in interleukin-3-containing medium. Conclusion We have established an SDS model cell line and have used this model to demonstrate that SBDS is not required for neutrophil maturation. However, SBDS knockdown cells were sensitive to apoptotic stimuli, indicating that SBDS acts to maintain survival of Granulocyte Precursor cells.
Kingo Fujimura - One of the best experts on this subject based on the ideXlab platform.
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knockdown of shwachman diamond syndrome gene sbds induces increased expression of galectin 1 and impaired cell growth
Blood, 2012Co-Authors: Masafumi Yamaguchi, Kingo Fujimura, Hanae Togayamaguchi, Hirokazu Kanegane, Naoki OkamuraAbstract:Abstract 2360 Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11. We have previously reported the Shwachman-Bodian-Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells which established by SBDS shRNAi were sensitive to apoptotic stimuli, and led to growth inhibition, indicating that SBDS acts to maintain survival of Granulocyte Precursor cells (Exp Hematol 35;579,2007). The precise mechanism by which the loss of SBDS inhibits growth of cells remains elusive. In order to clarify the impaired cell growth of SBDS knockdown cells, we analyzed two SDS patients (c.183_184TA>CT and c.258+2T>C) derived EB virus transformed lymphoblast cells (LCL). The growth of both LCL-SDS cell lines was considerably lower than control donor cells (LCL-C) which occurred in within 3 days of culture (1.4×10 6 cells/ml in LCL-C vs 5×10 5 cells/lm in LCL-SDS). LCL-C cells divided until 5 days, however, growth of LCL-SDS cells was saturated in 3days. When LCL-SDS cells were seeded to the fresh medium, LCL-SDS cells proliferated again. Conditional medium from 5 days SDS-LCL cell culture was then added to the culture of both LCL-C and LCL-SDS cell. This LCL-SDS conditional medium inhibited both LCL-C and LCL-SDS cell growth (50% and 60%, respectively), suggesting that growth inhibitors were secreted from LCL-SDS cells. In order to find growth inhibitors, we performed differential display. By annealing control primer based GeneFishing PCR screening, we found galectin-1 mRNA level was increased in LCL-SDS cells. We also confirmed that Galectin-1(Gal1) protein expression was markedly increased in LCL-SDS cells by western blot and conforcal microscopy. Gal1 was found to membrane bound, and it is plausible that Gal1 was secreted to the medium. In order to isolate Gal1 protein from medium, medium was passed through lactose agarose. Gal1 protein was purified from LCL-SDS cell culture medium, not from LCL-C cells. The inhibitory effect of Gal1 was confirmed using recombinant human Galectin-1 (rhGal1), which had similar dynamics to that of conditional medium from LCL-SDS cells. rhGal1 the proliferation of both LCL-C and LCL-SDS cells in a dose dependent manner. After exposure to rhGal1, Annexin V positive cells were increased in LCL-C cells (13.78±2.09% in control vs 16.83±2.81% in rhGal1, p=0.02). However, there was no difference in Gal1 induced apoptosis between LCL-C and LCL-SDS cells. In order to rescue growth failure of LCL-SDS cells, lactose, which modulates the binding between galectin and its substrate, was supplemented to the medium. Though lactose showed the growth inhibition of LCL cells, the viability of LCL-SDS cells was much higher than LCL-C cells. LCL-SDS cells were easily aggregated, however, the colony of LCL-SDS cell was much smaller in the presence of lactose. We also confirmed that Gal1 protein was overexpressed in SBDS knockdown 32Dcl3 cells, which were established by SBDS shRNAi. Conclusion: Overexpressed Gal1 was found from SDS patient9s derived LCL cells and SBDS shRNAi knockdown 32Dcl3 cells. Gal1 was also found in the conditional medium of LCL-SBDS cells, and secreted Gal1 inhibited the cell proliferation. These results indicated that Galectin-1 partially involved in growth failure of SBDS deficient cells. Disclosures: No relevant conflicts of interest to declare.
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mutations of the sbds gene result in nuclear mislocalization
Blood, 2008Co-Authors: Masafumi Yamaguchi, Kingo Fujimura, Naoki Okamura, Hanae Togayamaguchi, Valentina Svetic, Rajesh ChopraAbstract:Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11. We have previously reported that Shwachman-Bodian- Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells were sensitive to apoptotic stimuli, indicating that SBDS acts to maintain survival of Granulocyte Precursor cells. (Exp Hematol35; 579, 2007). A wide variety of mutations in SBDS gene has been identified, and almost of all patients show truncated immature proteins, p.K62X (c.183_184TA>CT) or p.C84fsX3 (c.258+2T>C). However, it is not yet clear how these truncated proteins affect cellular processes that result in the SDS phenotype. The SBDS protein is localized to the nucleoli but does not have the canonical nuclear localization signal. In order to clarify the molecular basis of pathogenicity of mutated SBDS proteins, we explored the subcellular distribution of normal and mutant SBDS proteins in Hela and 32Dcl3 cells. Using various N-terminal and C-terminal deletion constructs, we found N-terminal region, domain I (1-87 amino acid residue) in particular, was necessary to localize to the nucleus. The disease related mutations (C31W, K33E, N34I, L71P) and the mutations which are conserved among the species in the domain I (E44K, K62E, D70N, E82K) were generated. C31W and N34I mutants failed to localize SBDS to the nuclei. The SV40 derived nuclear localization signal was fused to these mutated SBDS protein, and these proteins were clearly localized to the nuclei. In addition to the mislocalization, the protein expression level of these mutants showed a dramatic decrease compared to the wild type. We also established SBDS wild type and domain I overexpressed 32Dcl3 cell. SBDS wild type overexpressed cells could differentiate to normal neutrophils in the presence of mG-CSF, however domain I overexpressed cells did not differentiate. Almost of all cells showed apoptosis in this domain I overexpressed cells in the presence of mG-CSF, and this was very similar like SBDS RNAi knockdown cells. The localization of endogenous SBDS protein was also analyzed in this domain I overexpressed cells. The domain I was concentrated to nuclei, however endogenous SBDS protein was diffused to cytosol. Conclusions: The present findings enable us to document the nuclear localization signals in SBDS domain I, and that the shuttling protein would promote SBDS to nuclei. These results also showed that mislocalization and/or low expression level of mutated SBDS protein would cause SDS.
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Shwachman-Diamond syndrome is not necessary for the terminal maturation of neutrophils but is important for maintaining viability of Granulocyte Precursors.
Experimental hematology, 2007Co-Authors: Masafumi Yamaguchi, Kingo Fujimura, Naoki Okamura, Hanae Toga, Asim Khwaja, Rajesh ChopraAbstract:Objective Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11, and disease-associated mutations were reported in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SBDS is a member of a highly conserved protein family in diverse species including archaea and eukaryotes. It is widely expressed in many tissues, and its function is still unknown. To investigate the function of the SBDS protein, we undertook loss-of-function experiments in the 32Dcl3 cell line, which has the potential to differentiate to mature neutrophils. Methods SBDS gene was downregulated with lentivirus-based RNAi system. SBDS knockdown cells were analyzed for surface marker expression by flow cytometry and analyzed for the sensitivity to apoptosis-inducing stimuli. Results After culture in Granulocyte colony-stimulating factor (G-CSF)-containing medium for 3 days, 32Dcl3 cells demonstrated normal proliferation but complete downregulation of SBDS protein expression. The SBDS RNAi knockdown cells did not proliferate in G-CSF-containing medium but after 7 days had the appearance of segmented neutrophils. The neutrophil maturation markers were detected on these cells. Undifferentiated SBDS RNAi knockdown cells demonstrated increased apoptosis of undifferentiated cells. Notably, SBDS RNAi knockdown cells demonstrated normal proliferation in interleukin-3-containing medium. Conclusion We have established an SDS model cell line and have used this model to demonstrate that SBDS is not required for neutrophil maturation. However, SBDS knockdown cells were sensitive to apoptotic stimuli, indicating that SBDS acts to maintain survival of Granulocyte Precursor cells.
Hanae Togayamaguchi - One of the best experts on this subject based on the ideXlab platform.
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knockdown of shwachman diamond syndrome gene sbds induces increased expression of galectin 1 and impaired cell growth
Blood, 2012Co-Authors: Masafumi Yamaguchi, Kingo Fujimura, Hanae Togayamaguchi, Hirokazu Kanegane, Naoki OkamuraAbstract:Abstract 2360 Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11. We have previously reported the Shwachman-Bodian-Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells which established by SBDS shRNAi were sensitive to apoptotic stimuli, and led to growth inhibition, indicating that SBDS acts to maintain survival of Granulocyte Precursor cells (Exp Hematol 35;579,2007). The precise mechanism by which the loss of SBDS inhibits growth of cells remains elusive. In order to clarify the impaired cell growth of SBDS knockdown cells, we analyzed two SDS patients (c.183_184TA>CT and c.258+2T>C) derived EB virus transformed lymphoblast cells (LCL). The growth of both LCL-SDS cell lines was considerably lower than control donor cells (LCL-C) which occurred in within 3 days of culture (1.4×10 6 cells/ml in LCL-C vs 5×10 5 cells/lm in LCL-SDS). LCL-C cells divided until 5 days, however, growth of LCL-SDS cells was saturated in 3days. When LCL-SDS cells were seeded to the fresh medium, LCL-SDS cells proliferated again. Conditional medium from 5 days SDS-LCL cell culture was then added to the culture of both LCL-C and LCL-SDS cell. This LCL-SDS conditional medium inhibited both LCL-C and LCL-SDS cell growth (50% and 60%, respectively), suggesting that growth inhibitors were secreted from LCL-SDS cells. In order to find growth inhibitors, we performed differential display. By annealing control primer based GeneFishing PCR screening, we found galectin-1 mRNA level was increased in LCL-SDS cells. We also confirmed that Galectin-1(Gal1) protein expression was markedly increased in LCL-SDS cells by western blot and conforcal microscopy. Gal1 was found to membrane bound, and it is plausible that Gal1 was secreted to the medium. In order to isolate Gal1 protein from medium, medium was passed through lactose agarose. Gal1 protein was purified from LCL-SDS cell culture medium, not from LCL-C cells. The inhibitory effect of Gal1 was confirmed using recombinant human Galectin-1 (rhGal1), which had similar dynamics to that of conditional medium from LCL-SDS cells. rhGal1 the proliferation of both LCL-C and LCL-SDS cells in a dose dependent manner. After exposure to rhGal1, Annexin V positive cells were increased in LCL-C cells (13.78±2.09% in control vs 16.83±2.81% in rhGal1, p=0.02). However, there was no difference in Gal1 induced apoptosis between LCL-C and LCL-SDS cells. In order to rescue growth failure of LCL-SDS cells, lactose, which modulates the binding between galectin and its substrate, was supplemented to the medium. Though lactose showed the growth inhibition of LCL cells, the viability of LCL-SDS cells was much higher than LCL-C cells. LCL-SDS cells were easily aggregated, however, the colony of LCL-SDS cell was much smaller in the presence of lactose. We also confirmed that Gal1 protein was overexpressed in SBDS knockdown 32Dcl3 cells, which were established by SBDS shRNAi. Conclusion: Overexpressed Gal1 was found from SDS patient9s derived LCL cells and SBDS shRNAi knockdown 32Dcl3 cells. Gal1 was also found in the conditional medium of LCL-SBDS cells, and secreted Gal1 inhibited the cell proliferation. These results indicated that Galectin-1 partially involved in growth failure of SBDS deficient cells. Disclosures: No relevant conflicts of interest to declare.
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mutations of the sbds gene result in nuclear mislocalization
Blood, 2008Co-Authors: Masafumi Yamaguchi, Kingo Fujimura, Naoki Okamura, Hanae Togayamaguchi, Valentina Svetic, Rajesh ChopraAbstract:Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The SDS disease locus was mapped to chromosome 7q11. We have previously reported that Shwachman-Bodian- Diamond syndrome (SBDS) gene is not required for neutrophil maturation. However, SBDS knockdown cells were sensitive to apoptotic stimuli, indicating that SBDS acts to maintain survival of Granulocyte Precursor cells. (Exp Hematol35; 579, 2007). A wide variety of mutations in SBDS gene has been identified, and almost of all patients show truncated immature proteins, p.K62X (c.183_184TA>CT) or p.C84fsX3 (c.258+2T>C). However, it is not yet clear how these truncated proteins affect cellular processes that result in the SDS phenotype. The SBDS protein is localized to the nucleoli but does not have the canonical nuclear localization signal. In order to clarify the molecular basis of pathogenicity of mutated SBDS proteins, we explored the subcellular distribution of normal and mutant SBDS proteins in Hela and 32Dcl3 cells. Using various N-terminal and C-terminal deletion constructs, we found N-terminal region, domain I (1-87 amino acid residue) in particular, was necessary to localize to the nucleus. The disease related mutations (C31W, K33E, N34I, L71P) and the mutations which are conserved among the species in the domain I (E44K, K62E, D70N, E82K) were generated. C31W and N34I mutants failed to localize SBDS to the nuclei. The SV40 derived nuclear localization signal was fused to these mutated SBDS protein, and these proteins were clearly localized to the nuclei. In addition to the mislocalization, the protein expression level of these mutants showed a dramatic decrease compared to the wild type. We also established SBDS wild type and domain I overexpressed 32Dcl3 cell. SBDS wild type overexpressed cells could differentiate to normal neutrophils in the presence of mG-CSF, however domain I overexpressed cells did not differentiate. Almost of all cells showed apoptosis in this domain I overexpressed cells in the presence of mG-CSF, and this was very similar like SBDS RNAi knockdown cells. The localization of endogenous SBDS protein was also analyzed in this domain I overexpressed cells. The domain I was concentrated to nuclei, however endogenous SBDS protein was diffused to cytosol. Conclusions: The present findings enable us to document the nuclear localization signals in SBDS domain I, and that the shuttling protein would promote SBDS to nuclei. These results also showed that mislocalization and/or low expression level of mutated SBDS protein would cause SDS.
