| Co-Investigator(Kenkyū-buntansha) |
YOKOYAMA Toshiiro Kyushu Univ., Fac.of Med., Physician, 医学部, 医員
ASANO Yoshinobu Kyushu Univ., Fac.of Med., Res.Assoc., 医学部, 助手 (60271110)
NAKASHIMA Hitoshi Kyushu Univ., Fac.of Med., Res.Assoc., 医学部, 助手 (70188960)
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| Research Abstract |
A.1.Detection of soluble G-CSF receptor
After incubation of human serum with biotinylated G-CSF,samples were subjected to native rplyacrylamide gel electrophorcsis (SDS-PAGE). Electrophoresed proteins were transferred to nitrocllulose membrane by electroblotting and peroxidase (PO) labeled streptavidin was reacted. PO reaction reveled the existence of G-CSF binding substances. Two molecules were detected, one was 95kD and the other was 105kD.After subtracting 20kD of G-CSF itself, these molecules were 75kd and 85kd, respectively. Furthermore, we developed antibodies against the polypeptides of various domains of G-CSF receptor protein deduced by cDNA structure. Similar results were obtained using these antitibodies and these findings indicate the existence of soluble G-CSF receptor at physiological condition.
B.Structure of soluble G-CSF receptor
Expression states of G-CSF receptor on various maturation states of neutrophilic graunlocytes were studies with reverse transcription polymerase
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chain reactions (RT-PCR). Since soluble G-CSF receptor was probably coded by type II G-CSF receptor cDNA (formed by alternative splicing), we used the primers to encompass whole transmembrane domain to detect type II G-CSF receptor cDNA (truncated type) as well as type I (wild type, full length) G-CSF receptor cDNA as different size bands. We fractionated bone marrow neutrophil lineage cells with flowcytometric cell sorter to 4 maturation stage cell populations and found the intensity of small molecular weight band (soluble G-CSF receptor) increased during maturation stages.
C.Action of soluble G-CSF receptor
Action of soluble murine G-CSF receptor which was made from the cDNA transfected E.coli was examined. When it was added to human bone marrow cultue in vitro, the formation of granulocyte colony was significantly suppressed. Human myeloid leukemic cell proliferation was also suppressed by this solublized murine G-CSF.Furthermore, we found new type soluble G-CSF receptor from a patient with severe congenital neutropenia. This new variant also detected in human myeloid leukemia cell lines. While severe suppression of normal granulopoiesis is often observed in patients with acute myeloblastic leukemia, the variant form of soluble G-CSF receptor may possess strong proliferation suppressing activity to normal granulopoiesis but leukemic cells. To prove this hypothesis, the new variant form of soluble G-CSF receptor cDNA has been expressed in mammalian CHO cells. Less
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