Analysis of the mutant β-actin functions in the mutant actin transduced mouse

我们报告一位12岁女性患者，患有突变型β-肌动蛋白。她的实验室分析显示血小板减少症、白细胞减少症和中性粒细胞功能障碍。该β-肌动蛋白突变位点位于profilin的结合位点。基于这些研究，我们发现突变的肌动蛋白作为显性负性遗传发挥作用，并参与中性粒细胞功能障碍，如趋化性降低和超氧化物产生减少（PNAS 1999）。到目前为止，已经报道了中性粒细胞肌动蛋白功能障碍（NAD）和具有异常47-和89-kDa蛋白的NAD（NAD 47/89）的病例。最近，我们从分子水平分析了一例Rac 2突变导致中性粒细胞趋化功能障碍的病例。我们将这4例病例作为中性粒细胞骨架疾病进行回顾，因为患者的中性粒细胞功能障碍是显著的，没有明显的全身症状（中性粒细胞骨架疾病）。Int J Hematol 74：119- 24，2001）在本项目中，我们试图获得表达该突变型β-actin的K562细胞系，并对该突变型β-actin进行分析。 关于我们 - 肌动蛋白功能。但是这种突变肌动蛋白的表达量很小，我们无法检测到任何抑制活性。虽然我们也尝试用突变型β-actin Knock-in小鼠克隆ES细胞系，但在本项目中未能获得。我们与本论文的合著者Yamasaki博士合作，仍在尝试获得突变型β-actin基因敲入小鼠。使用这些为β-actin项目开发的技术，我们发现了以下有关中性粒细胞超氧化物产生的事实。1)在与Sumimoto博士的合作中，我们克隆了与gp 91-phox，p47 phox和p67 phox同源的新蛋白质的人cDNA，命名为Nox 5，p41 nox和p51 nox，它们分别是NADPH氧化酶的催化和调节蛋白。(EMBO J. 21：6312- 20，2002，JBC 2003出版）。2)与Sugimoto博士合作，小鼠移植的骨髓细胞转导了双顺反子逆转录病毒载体Ha-MDR-IRES-gp 91，在外周血单核细胞中显示20-30%的P-糖蛋白和人gp 91共表达超过1年。这些结果表明，人多药抗性基因（MDR 1）与治疗基因的共表达为遗传修饰的细胞提供了可选择的生长优势（J Gene Med vol.4，2003）。少

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 … More -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