|Budget Amount *help
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
We reported a 12 year-old female patient with mutant β-actin. Her laboratory analysis showed thrombocytopenia, leukopenia and neutrophil dysfunctions. This β-actin mutation site was at a binding site for a profilin. Based on these studies, we showed the mutant actin work as a dominant negative inheritance and involved in the neutrophile dysfunctions, such as decreased chemotaxis and superoxide production (PNAS 1999). So far, cases of neutrophil actin dysfunction (NAD) and NAD with abnormal 47- and 89-kDa proteins (NAD 47/89) have been reported. Recently, a case with Rac 2 mutation that had neutrophil chemotactic dysfunction, were analyzed at the molecular level. We review these 4 cases as neutrophil cytoskeletal disease, because neutrophil dysfunctions of the patients are remarkable without an apparent systemic symptoms (Neutrophile cytoskeletal disease. Int J Hematol 74 : 119-24,2001)
In this project, we tried to get this mutant β-actin expressed K562 cell line and analyze this mutantβ
-actin function. But this mutant actin protein was expressed so small amount that we could not detect any inhibitory activity. Although we also tried to clone ES cell line with the mutantβ-actin Knock-in mouse, we could not get it during this project. Collaborating with Dr. Yamasaki who is the coauthor of our paper, we are still trying to get the mutantβ-actin knock-in mouse.
Using in these technique developed forβ-actin project, we found the following facts concerning about neutrophile superoxide production. 1) In a collaboration with Dr. Sumimoto, we cloned human cDNAs for novel proteins homologous to gp91-phox, p47phox and p67phox, designated Nox 5, p41nox and p51nox, those are the catalytic and regulatory proteins of NADPH oxidase, respectively. (EMBO J. 21 : 6312-20,2002, JBC 2003 in press). 2) Collaborating with Dr. Sugimoto, mice transplanted bone marrow cells that was transduced a bicistronic retrovirus vector, Ha-MDR-IRES-gp91, showed 20-30% co-expression of P-glycoprotein and human gp91 in peripheral blood mononuclear cells for over 1 year. These results suggest that co-expression of a human multidrug resistance gene (MDR1) with a therapeutic gene affords selectable growth advantage to genetically modified cells (J Gene Med vol.4, 2003). Less