Analysis of molecular mechanisms by which heme oxygenase-carbon monoxide pathway regulates cellular functions under physiological and pathophysiological conditions using newly developed gene targeting mice.

使用新开发的基因靶向小鼠分析血红素加氧酶-一氧化碳途径在生理和病理生理条件下调节细胞功能的分子机制。

基本信息

  • 批准号:
    14370063
  • 负责人:
  • 金额:
    $ 7.17万
  • 依托单位:
  • 依托单位国家:
    日本
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
  • 财政年份:
    2002
  • 资助国家:
    日本
  • 起止时间:
    2002 至 2004
  • 项目状态:
    已结题

项目摘要

Heme oxygenase (HO), an enzyme capable to degrade protoheme IX into carbon monoxide (CO), free divalent iron, and biliverdin-IXα, plays important roles in the regulation of many cellular functions. However, the detailed molecular mechanisms of HO-dependent biological functions remain elusive. In this study, we aimed to establish new HO gene-targeting mice to address isozyme-specific roles, to find new targets of endogenous CO, and to examine effects of CO and other gaseous molecules such as nitric oxide (NO) on the same targeting molecules in vivo.To circumvent embryonic lethality and infertility caused by an inactivation of the HO-1 gene, we constructed a new targeting vector containing exon2 of the gene flanked by loxP sequences, which can be deleted by Cre recombinase in a tissue-specific manner in vivo, in order to generate conditional HO-1 knockout mice. Two distinct ES cell clones, in which an appropriate homologous recombination occurred, were obtained so far, and chimeric mice … More have been generated.To find novel targeting molecules of CO, we examined alterations of metabolites in mouse liver treated with acetaminophen (AAP), by which intrahepatic HO activity and consequent CO production were increased, using CE-MS. We found that the levels of S-adenosylmethionine, cysteine, glutathione, and taurine were significantly reduced by AAP treatment. On the other hand, great accumulation of methionine occurred in the affected liver. These results suggest that CO might suppress the flux into a transsulfuration pathway by binding to and inhibiting cystathionine β-synthase (CBS), a rate-limiting enzyme of the pathway. We next examined how CO inhibits CBS activity using the partially purified enzyme in vitro. Our findings showed that mili molar levels of CO could cleave the Fe of heme-cysteine bond and form a carboxy-heme complex, resulting in an inhibition of the enzyme activity.To clarify the roles of CO and NO to regulate soluble guanylate cyclase (sGC) in vivo, we carried out immunohistochemical analyses to visualize the gas-mediated sGC activation by suppressing endogenous these gaseous molecules separately. Under conditions where CO was suppressed by ZnPP, all retinal cell layers homogenously exhibited enhancement of NO-dependent activation of sGC. On the other hand, under NO-suppressing conditions, eliminating CO abrogated the basal sGC activation in a layer-specific manner. These results suggest that endogenous CO plays a role in fine tuning the NO-dependent activation of sGC function by suppressing the maximum response as well as by modestly elevating the minimum response in particular layer of retina. Less
血红素加氧酶(HO)是一种能够将血红素IX降解为一氧化碳(CO)、游离二价铁和胆绿素IX α的酶,在许多细胞功能的调节中起重要作用。然而,HO依赖性生物学功能的详细分子机制仍然难以捉摸。在这项研究中,我们的目的是建立新的HO基因靶向小鼠,以解决同工酶特异性作用,寻找内源性CO的新靶点,并检查CO和其他气体分子如一氧化氮(NO)对体内相同靶向分子的影响。我们构建了一个新的靶向载体,该载体含有基因的外显子2,侧翼是loxP序列,该序列可以在体内以组织特异性方式被Cre重组酶删除,以产生条件性HO-1敲除小鼠。到目前为止,获得了两个不同的ES细胞克隆,其中发生了适当的同源重组, ...更多信息 为了找到新的靶向分子的CO,我们研究了改变代谢物在小鼠肝脏治疗对乙酰氨基酚(AAP),通过肝内HO活性和随之产生的CO生产增加,使用CE-MS。我们发现,S-腺苷甲硫氨酸,半胱氨酸,谷胱甘肽和牛磺酸的水平显着降低AAP治疗。另一方面,在受影响的肝脏中发生了大量的蛋氨酸积累。这些结果表明,CO可能通过结合并抑制硫代胱硫醚β-合成酶(CBS)来抑制转硫途径的流量,CBS是转硫途径的限速酶。接下来,我们使用部分纯化的酶在体外研究了CO如何抑制CBS活性。我们的研究结果表明,微摩尔水平的CO可以切割血红素-半胱氨酸键的Fe,形成羧基-血红素复合物,导致酶活性的抑制.为了阐明CO和NO在体内调节可溶性鸟苷酸环化酶(sGC)的作用,我们进行了免疫组织化学分析,以显示气体介导的sGC激活通过抑制内源性这两种气体分子分别.在CO被ZnPP抑制的条件下,所有视网膜细胞层均表现出增强的sGC的NO依赖性激活。另一方面,在NO抑制条件下,消除CO以层特异性方式废除了基础sGC活化。这些结果表明,内源性CO通过抑制最大响应以及通过适度升高视网膜特定层中的最小响应,在微调sGC功能的NO依赖性激活中起作用。少

项目成果

期刊论文数量(79)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Suppressed apoptosis in the inflamed gastric mucosa of Helicobacter pylori-colonized iNOS-knockout mice.
  • DOI:
    10.1016/s0891-5849(03)00218-1
  • 发表时间:
    2003-06
  • 期刊:
  • 影响因子:
    7.4
  • 作者:
    M. Miyazawa;Hidekazu Suzuki;T. Masaoka;A. Kai;M. Suematsu;H. Nagata;S. Miura;H. Ishii
  • 通讯作者:
    M. Miyazawa;Hidekazu Suzuki;T. Masaoka;A. Kai;M. Suematsu;H. Nagata;S. Miura;H. Ishii
Role of thromboxane derived from COX-1 and -2 in hepatic microcirculatoyr dysfunction during endotoxemia in mice.
COX-1和-2衍生的血栓素在小鼠内毒素血症期间肝微循环功能障碍中的作用。
  • DOI:
  • 发表时间:
    2004
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Katagiri;H.
  • 通讯作者:
    H.
Nagano, S: "Infrared spectroscopic and mutational studies on putidaredoxin-induced conformational changes in ferrous CO-P450cam."Biochemistry. 42・49. 14507-14514 (2003)
Nagano, S:“恶臭氧还蛋白诱导的亚铁 CO-P450cam 构象变化的红外光谱和突变研究”。生物化学 42・49 (2003)。
  • DOI:
  • 发表时间:
  • 期刊:
  • 影响因子:
    0
  • 作者:
  • 通讯作者:
Mechanistic probing of gaseous signal transduction in microcirculation.
  • DOI:
    10.1089/152308603768295230
  • 发表时间:
    2003-08
  • 期刊:
  • 影响因子:
    6.6
  • 作者:
    M. Suematsu;Kazuhiro Suganuma;S. Kashiwagi
  • 通讯作者:
    M. Suematsu;Kazuhiro Suganuma;S. Kashiwagi
Infrared spectroscopic and mutational studies on putidaredoxin-induced conformational changes in ferrous CO-P450cam
  • DOI:
    10.1021/bi035410p
  • 发表时间:
    2003-12-16
  • 期刊:
  • 影响因子:
    2.9
  • 作者:
    Nagano, S;Shimada, H;Ishimura, Y
  • 通讯作者:
    Ishimura, Y
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SUEMATSU Makoto其他文献

SUEMATSU Makoto的其他文献

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{{ truncateString('SUEMATSU Makoto', 18)}}的其他基金

Biomedical Application of Gas Biology through Multidisciplinary Approaches
通过多学科方法进行气体生物学的生物医学应用
  • 批准号:
    17GS0419
  • 财政年份:
    2005
  • 资助金额:
    $ 7.17万
  • 项目类别:
    Grant-in-Aid for Creative Scientific Research
Development of artificial oxygen carriers under consideration of microvascular homeostasis
考虑微血管稳态的人工氧载体的开发
  • 批准号:
    13557132
  • 财政年份:
    2001
  • 资助金额:
    $ 7.17万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
HEME OXYGENASE-1-MEDIATED STRESS RESPONSE OF LIVER AND RETICULOENDOTHELIAL SYSTEM
血红素加氧酶-1介导的肝脏和网状内皮系统应激反应
  • 批准号:
    12470128
  • 财政年份:
    2000
  • 资助金额:
    $ 7.17万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Roles of Carbon monoxide in regulation of sinusoidal blood flow and bile excretion
一氧化碳在调节血窦血流和胆汁排泄中的作用
  • 批准号:
    09470143
  • 财政年份:
    1997
  • 资助金额:
    $ 7.17万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
CO as a novel signaling molecule modulating cell and organ function.
CO 作为调节细胞和器官功能的新型信号分子。
  • 批准号:
    08044318
  • 财政年份:
    1996
  • 资助金额:
    $ 7.17万
  • 项目类别:
    Grant-in-Aid for international Scientific Research
Roles of low molecular ononoxides in regulation of hepatobiliary function
低分子氧化氮在肝胆功能调节中的作用
  • 批准号:
    06454264
  • 财政年份:
    1994
  • 资助金额:
    $ 7.17万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (B)
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