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Articles by Y Usami
Total Records ( 2 ) for Y Usami
  N Ishimine , Y Usami , S Nogi , T Sumida , Y Kurihara , K Matsuda , K Nakamura , K Yamauchi , N Okumura and M. Tozuka
  Background

In human serum, a portion of homocysteine (Hcy) exists as an N-linked form to the -amino group of protein lysine residues. N-homocysteinylated proteins differ structurally and functionally from native proteins. The present study strives to develop detection and potential semi-quantification methods for N-homocysteinylated apolipoprotein AI (N-Hcy-apoAI) in human serum.

Methods

Serum treated with or without cysteamine was supplied to isoelectric focusing (IEF) followed by an immunoblot using an anti-apoAI antibody. Cysteamine treatment increased the isoelectric point for N-Hcy-apoAI, but not for unmodified apoAI, due to the presence of -SH group(s) derived from Hcy and the absence of a cysteine residue in the apoAI molecule. N-Hcy-apoAI was semi-quantified from the scanned immunoblot pattern via a computer.

Results

After cysteamine treatment, N-Hcy-apoAI in the serum was identified by IEF at the position with a higher pI value compared with intact apoAI. The reproducibility (between assays) of the semi-quantification method was 19.1% CV (coefficient of variation) for an average ratio 5.9% of N-Hcy-apoAI to the whole apoAI in the serum. Approximately 1.0–7.4% of apoAI was N-homocysteinylated in the serum obtained from 27 healthy subjects. Neither the ratio of N-Hcy-apoAI nor its concentration, calculated by total apoAI concentration, indicated correlation with the so-called total (free and S-linked) Hcy concentration.

Conclusions

We directly found that a portion of apoAI in the serum undergoes homocysteinylation in an N-linkage manner, and used this to develop a potential semi-quantification method for N-Hcy-apoAI.

  T Yazawa , Y Inaoka , R Okada , T Mizutani , Y Yamazaki , Y Usami , M Kuribayashi , M Orisaka , A Umezawa and K. Miyamoto
 

Previously, we demonstrated that bone marrow-derived mesenchymal stem cells (MSCs) differentiate into steroidogenic cells such as Leydig and adrenocortical cells by the introduction of steroidogenic factor-1 (SF-1) and treatment with cAMP. In this study, we employed the same approach to differentiate umbilical cord blood (UCB)-derived MSCs. Despite UCB-MSCs differentiating into steroidogenic cells, they exhibited characteristics of granulosa-luteal-like cells. We found that peroxisome proliferator-activated receptor- coactivator-1 (PGC-1) was expressed and further induced by cAMP stimulation in UCB-MSCs. Consistent with these results, tissue-specific expression of Pgc-1 was observed in rat ovarian granulosa cells. PGC-1 binds to the NR5A family [SF-1 and liver receptor homolog-1 (LRH-1)] of proteins and markedly enhances their transcriptional activities. Reporter assays revealed that PGC-1 activated the promoter activities of SF-1 and LRH-1 target genes. Infection of KGN cells (a human cell line derived from granulosa cells) with adenoviruses expressing PGC-1 resulted in the induction of steroidogenesis-related genes and stimulation of progesterone production. PGC-1 also induced SF-1 and LRH-1, with the latter induced to a greater extent. Knockdown of Pgc-1 in cultured rat granulosa cells resulted in attenuation of gene expression as well as progesterone production. Transactivation of the NR5A family by PGC-1 was repressed by Dax-1. PGC-1 binds to the activation function 2 domain of NR5A proteins via its consensus LXXLL motif. These results indicate that PGC-1 is involved in progesterone production in ovarian granulosa cells by potentiating transcriptional activities of the NR5A family proteins.

 
 
 
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