| Project/Area Number |
11670992
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Hematology
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| Research Institution | Mie University |
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Principal Investigator |
NISHIKAWA Masakatsu Mie University, Hospital, lecturer, 医学部・附属病院, 講師 (30144257)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | Nuclear lobulation / NB4 cells / granulocytic differentiation / myosin II / all trans retinoic acid / Rho-kinase / myosin phosphatase / phosphorylated myosin light chain / アクチン / HL-60細胞 |
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| Research Abstract |
Nuclearlobulation may enhance neutrophil deformability and movement through vessel walls and into sites of inflammation. However the mechanism of nuclear segmentation during granulocytic differentiation remains unknown. Two principal isoforms of non-muscle myosin II, A and B (MHC-IIA and MHC-IIB) are present in different proportions in different tissues. Activity of myosin II is functionally regulated through phosphorylation of 20kD light chain of myosin (MLC_<20>) at Ser19 by MLC kinase and Rho-kinase. We determined whether non-muscle myosin II has a functional role in nuclear lobulation during all trans retinoic acid (ATRA)-induced granulocytic differentiation of human promyelocytic leukemia cell line, NB4 cells. Isoform-specific polyclonal antibodies reveal the cellular distributions of these isoforms in interphase and in dividing NB4 cells. NB4 cells expressed mostly MHC-IIA and a small amount of MHC-IIB.The level of MHC-IIA was increased during ATRA-induced granulocytic differentiation, while that of MHC-IIB was unaltered. Expressions of Rho-kinase and myosin phosphatase (e.g.myosin-binding subunit ) were unchanged during ATRA-induced NB4 cell differentiation. Immunofluorescence confocal microscopy demonstrated distinct localizations for MHC-IIA and MHC-IIB in wild NB4 cells. In interphase NB4 cells, MHC-IIB was present in the cell cortex and diffusely arranged in the cytoplasm, while MHC-IIA was localized in perinuclear region. MHC-IIA and phosphorylated MLC_<20> at Ser19 were colocalized to the deavage furrows (contractile ring) in dividing NB4 cells. In ATRA-induced granulocytes of NB4 cells, MHC-IIA and phosphorylated MLC_<20> were colocalized to the bridging area between two nuclear lobes. Rho-kinase and myosin phosphatase also colocalized to this area. These results suggest that MHC-IIA has a important role in nuclear lobulation during ATRA-induced granulocytic differentiation of NB4 cells.
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