Earticle

초록

영어

Mitochondria diseases have been reported to involve structural and functional defects of complex I-V. Especially, many of these diseases are known to be related to dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I). The dysfunction of mitochondria complex I is associated with neurodegenerative disorders, such as Parkinson's disease, Huntington's disease, and Leber’s hereditary optic neuropathy (LHON).
Mammalian mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) is largest and consists of at least 46 different subunits. In contrast, the NDI1 gene of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. The Saccharomyces cerevisiae NDI1 gene using a recombinant adeno-associated virus vector (rAAV-NDI1) was successfully expressed in AML12 mouse liver hepatocytes and the NDI1-transduced cells were able to grow in media containing rotenone. In contrast, control cells that did not receive the NDI1 gene failed to survive. The expressed Ndi1 enzyme was recognized to be localized in mitochondria by confocal immunofluorescence microscopic analyses and immunoblotting.
Using digitonin-permeabilized cells, it was shown that the NADH oxidase activity of the NDI1-transduced cells was not affected by rotenone which is inhibitor of complex I, but was inhibited by antimycin A. Furthermore, these results indicate that Ndi1 can be functionally expressed in the AML12 mouse liver hepatocytes. It is conceivable that the NDI1 gene is powerful tool for gene therapy of mitochondrial diseases caused by complex I deficiency. In the future, we will attempt to functionally express the NDI1 gene in mouse embryonic stem (mES) cell.

목차

ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
  Preparation of rAAV-NDI1
  Cell Culture and rAAV-NDI1 Infection
  Viability Measurements
  Immunofluorescence
  Isolation of Mitochondria and Mitochondrial Membrane Fraction
  O2 Consumption Measurements
  Other Analytical Procedures
  Materials
  Statistical Analysis
 RESULTS
  Functional Expression of the NDI1 Gene in AML12 Mouse Liver Hepatocytes
  Effect of the Ndi1 Expression on the Electron Transfer Activity
  Western blotting Result of the Yeast NDI1 in AML12 Cells
  Effects of Complex I Inhibitors on Cell Growth
 DISCUSSION
 REFERENCES

키워드

저자

• Byoung Boo Seo [ Dept. of Animal Resources, College of Life & Environmental Science, Daegu University ]
• Humdai Park [ Dept. of Biotechnology, College of Engineering, Daegu University ] Corresponding author

참고문헌

발행기관

간행물

표지보기 관심저널 등록

• 간행물명

  Reproductive & developmental biology

• 간기

  계간

• pISSN

  1738-2432

• eISSN

  2288-0151

• 수록기간

  1977~2018

• 십진분류

  KDC 527 DDC 636