2000 Fiscal Year Final Research Report Summary
The Glycation as an Etiology of Diabetic Microangiopathy.-Gene-targeting of the Enzyme Metabolizing Glycation Intermediates.-
Project/Area Number |
11671116
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Research Category |
Grant-in-Aid for Scientific Research (C)
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Allocation Type | Single-year Grants |
Section | 一般 |
Research Field |
Metabolomics
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Research Institution | Kobe University |
Principal Investigator |
SATOSHI Miyata Kobe University School of medicine, assistant professor, 医学部, 助手 (20304090)
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Project Period (FY) |
1999 – 2000
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Keywords | Diabetic complications / microangiopathy / glycation / 3-deoxyglucosone / dicarbonyl compound / aldehyde reductase / gene targeting |
Research Abstract |
It is known that the formation of highly reactive dicarbonyl compounds such as 3-deoxyglucosone (3-DG), an intermediate of the glycation reaction, is accelerated in diabetic subjects. Several lines of evidence have shown that excess 3-DG influences cell functions in vitro, suggesting its involvement in the development of diabetic microangiopathy. On the other hand, it has been proposed that there is a defense system against 3-DG by metabolizing it to an inert agent in vivo. In this regard, previous studies have shown that aldehyde reductase (ALR) plays a major role in it. Thus, the present study was designed to clarify the relationship between 3-DG metabolism and alteration in microvascular tissues by establishing ALR-knockout mice. We first prepared a probe to clone mouse ALR cDNA by conducting PCR on mouse liver cDNA library with a set of primer deduced from the known rat ALR cDNA sequence. Using this probe, we successfully cloned a full length of mALR cDNA in 1999. We subsequently obtained a genome ALR DNA from a mouse genome DNA library with a probe including the start codon, followed by analysis of its sequence. We then constructed a targeting vector aiming homologous replacement in its exon 2〜5. Simultaneously, we prepared certain probes to subsequently screen gene-targeted ES cells by Southern blotting among the transfected cells with the vector described above. We are going to microinject the ALR-gene-targeted ES cells into early embryo for generating chimeras, followed by establishing knockout mice. Finally, the mice were analyzed in terms of the relationship between 3-DG metabolism and alterations occurring in microvasculature tissues.
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