The Experts below are selected from a list of 27 Experts worldwide ranked by ideXlab platform
Koji Owada - One of the best experts on this subject based on the ideXlab platform.
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Differential activation and redistribution of c-Src and Fyn in platelets, assessed by MoAb specific for C-terminal tyrosine-dephosphorylated c-Src and Fyn.
Biochimica et Biophysica Acta, 2000Co-Authors: Yi Wu, Yukio Ozaki, Katsue Inoue, Kaneo Satoh, Tsukasa Ohmori, Yutaka Yatomi, Koji OwadaAbstract:Abstract Tyrosine kinases, c-Src and Fyn, in their active form, have their C-terminal tyrosine residue dephosphorylated. In this study, we used clone 28, a monoclonal antibody (MoAb) that recognizes dephosphorylated C-terminal tyrosine of c-Src and Fyn, to investigate the mode of activation and mobilization of these kinases. Independently of integrin αIIbβ3 signaling, the Fyn activity increased by 8.3-fold 5 s after stimulation with 20 μM TRAP (thrombin receptor agonist peptide), while that of c-Src increased only by 2.9-fold 15 s after stimulation. Both c-Src and Fyn translocated to the Triton-insoluble cytoskeletal fraction in an aggregation-dependent manner. Five minutes after TRAP-stimulation, 85% of Fyn translocated to the cytoskeleton, while only about 20% of c-Src was recovered in this fraction. The Triton-insoluble fraction was further fractionated by RIPA (Radioimmunoprecipitation Assay) Buffer containing 0.1% SDS. While active c-Src was predominantly present in the Triton-insoluble/RIPA-insoluble fraction, clone 28-negative c-Src was present in the Triton-insoluble/RIPA-soluble fraction. On the other hand, Fyn was present only in the Triton-insoluble/RIPA-insoluble fraction. These findings suggest that the mode of activation and redistribution into the cytoskeleton differs between c-Src and Fyn, and that clone 28 provides a useful tool for investigating the activation and mobilization of Src family tyrosine kinases.
Takahiro Kusakabe - One of the best experts on this subject based on the ideXlab platform.
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Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System
Molecular Biotechnology, 2016Co-Authors: Kazuhiro Iiyama, Tuneyuki Tatsuke, Takahiro KusakabeAbstract:Baculovirus - Bombyx mori protein expression system has mainly been used for translation of eukaryotic proteins. In contrast, information pertaining to bacterial protein expression using this system is not sufficient. Therefore, recombinant nucleases from Serratia liquefaciens (rSlNucAs) were expressed in a Baculovirus - B. mori protein expression system. rSlNucAs containing the native signal peptide (rSlNucA-NSP) or silkworm 30-K signal peptide (rSlNucA-30K) at the NH_2-terminus were constructed to enable secretion into the extracellular fraction. Both rSlNucA-30K and rSlNucA-NSP were successfully secreted into hemolymph of B. mori larvae. Affinity-purified rSlNucAs showed high nuclease activity. Optimum pH was 7.5 and half of maximum activity was maintained between pH 7.0 and 9.5. Optimum temperature was 35 °C. rSlNucAs showed sufficient activity in twofold-diluted Radioimmunoprecipitation Assay Buffer and undiluted, mild lysis Buffer. Genomic DNA of Escherichia coli was efficiently digested by rSlNucAs in the bacterial lysate. The results in this study suggest that rSlNucAs expressed by the Baculovirus - B. mori protein expression system will be a useful tool in molecular biology. Functional recombinant protein of bacteria was produced by Baculovirus - B. mori protein expression system. This system may be highly suitable for bacterial extracellular protein secreted via Sec pathway.
Yi Wu - One of the best experts on this subject based on the ideXlab platform.
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Differential activation and redistribution of c-Src and Fyn in platelets, assessed by MoAb specific for C-terminal tyrosine-dephosphorylated c-Src and Fyn.
Biochimica et Biophysica Acta, 2000Co-Authors: Yi Wu, Yukio Ozaki, Katsue Inoue, Kaneo Satoh, Tsukasa Ohmori, Yutaka Yatomi, Koji OwadaAbstract:Abstract Tyrosine kinases, c-Src and Fyn, in their active form, have their C-terminal tyrosine residue dephosphorylated. In this study, we used clone 28, a monoclonal antibody (MoAb) that recognizes dephosphorylated C-terminal tyrosine of c-Src and Fyn, to investigate the mode of activation and mobilization of these kinases. Independently of integrin αIIbβ3 signaling, the Fyn activity increased by 8.3-fold 5 s after stimulation with 20 μM TRAP (thrombin receptor agonist peptide), while that of c-Src increased only by 2.9-fold 15 s after stimulation. Both c-Src and Fyn translocated to the Triton-insoluble cytoskeletal fraction in an aggregation-dependent manner. Five minutes after TRAP-stimulation, 85% of Fyn translocated to the cytoskeleton, while only about 20% of c-Src was recovered in this fraction. The Triton-insoluble fraction was further fractionated by RIPA (Radioimmunoprecipitation Assay) Buffer containing 0.1% SDS. While active c-Src was predominantly present in the Triton-insoluble/RIPA-insoluble fraction, clone 28-negative c-Src was present in the Triton-insoluble/RIPA-soluble fraction. On the other hand, Fyn was present only in the Triton-insoluble/RIPA-insoluble fraction. These findings suggest that the mode of activation and redistribution into the cytoskeleton differs between c-Src and Fyn, and that clone 28 provides a useful tool for investigating the activation and mobilization of Src family tyrosine kinases.
Kazuhiro Iiyama - One of the best experts on this subject based on the ideXlab platform.
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Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System
Molecular Biotechnology, 2016Co-Authors: Kazuhiro Iiyama, Tuneyuki Tatsuke, Takahiro KusakabeAbstract:Baculovirus - Bombyx mori protein expression system has mainly been used for translation of eukaryotic proteins. In contrast, information pertaining to bacterial protein expression using this system is not sufficient. Therefore, recombinant nucleases from Serratia liquefaciens (rSlNucAs) were expressed in a Baculovirus - B. mori protein expression system. rSlNucAs containing the native signal peptide (rSlNucA-NSP) or silkworm 30-K signal peptide (rSlNucA-30K) at the NH_2-terminus were constructed to enable secretion into the extracellular fraction. Both rSlNucA-30K and rSlNucA-NSP were successfully secreted into hemolymph of B. mori larvae. Affinity-purified rSlNucAs showed high nuclease activity. Optimum pH was 7.5 and half of maximum activity was maintained between pH 7.0 and 9.5. Optimum temperature was 35 °C. rSlNucAs showed sufficient activity in twofold-diluted Radioimmunoprecipitation Assay Buffer and undiluted, mild lysis Buffer. Genomic DNA of Escherichia coli was efficiently digested by rSlNucAs in the bacterial lysate. The results in this study suggest that rSlNucAs expressed by the Baculovirus - B. mori protein expression system will be a useful tool in molecular biology. Functional recombinant protein of bacteria was produced by Baculovirus - B. mori protein expression system. This system may be highly suitable for bacterial extracellular protein secreted via Sec pathway.
Yutaka Yatomi - One of the best experts on this subject based on the ideXlab platform.
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Differential activation and redistribution of c-Src and Fyn in platelets, assessed by MoAb specific for C-terminal tyrosine-dephosphorylated c-Src and Fyn.
Biochimica et Biophysica Acta, 2000Co-Authors: Yi Wu, Yukio Ozaki, Katsue Inoue, Kaneo Satoh, Tsukasa Ohmori, Yutaka Yatomi, Koji OwadaAbstract:Abstract Tyrosine kinases, c-Src and Fyn, in their active form, have their C-terminal tyrosine residue dephosphorylated. In this study, we used clone 28, a monoclonal antibody (MoAb) that recognizes dephosphorylated C-terminal tyrosine of c-Src and Fyn, to investigate the mode of activation and mobilization of these kinases. Independently of integrin αIIbβ3 signaling, the Fyn activity increased by 8.3-fold 5 s after stimulation with 20 μM TRAP (thrombin receptor agonist peptide), while that of c-Src increased only by 2.9-fold 15 s after stimulation. Both c-Src and Fyn translocated to the Triton-insoluble cytoskeletal fraction in an aggregation-dependent manner. Five minutes after TRAP-stimulation, 85% of Fyn translocated to the cytoskeleton, while only about 20% of c-Src was recovered in this fraction. The Triton-insoluble fraction was further fractionated by RIPA (Radioimmunoprecipitation Assay) Buffer containing 0.1% SDS. While active c-Src was predominantly present in the Triton-insoluble/RIPA-insoluble fraction, clone 28-negative c-Src was present in the Triton-insoluble/RIPA-soluble fraction. On the other hand, Fyn was present only in the Triton-insoluble/RIPA-insoluble fraction. These findings suggest that the mode of activation and redistribution into the cytoskeleton differs between c-Src and Fyn, and that clone 28 provides a useful tool for investigating the activation and mobilization of Src family tyrosine kinases.
