Keap1 and Cul3 constitute a unique ubiquitin E3 ligase that degrades Nrf2, a key activator of cytoprotective genes. Upon exposure to oxidants/electrophiles, the enzymatic activity of this ligase complex is inhibited and the complex fails to degrade Nrf2, resulting in the transcriptional activation of Nrf2 target genes. Keap1 possesses several reactive cysteine residues that covalently bond with electrophiles in vitro. To clarify the functional significance of each Keap1 cysteine residue under physiological conditions, we established a transgenic complementation rescue model. The transgenic expression of mutant Keap1(C273A) and/or Keap1(C288A) protein in Keap1 null mice failed to reverse constitutive Nrf2 activation, indicating that cysteine residues at positions 273 and 288 are essential for Keap1 to repress Nrf2 activity in vivo. In contrast, Keap1(C151S) retained repressor activity and mice expressing this molecule were viable. Mouse embryonic fibroblasts from Keap1(C151S) transgenic mice displayed decreased expression of Nrf2 target genes both before and after an electrophilic challenge, suggesting that Cys151 is important in facilitating Nrf2 activation. These results demonstrate critical roles of the cysteine residues in vivo in maintaining Keap1 function, such that Nrf2 is repressed under quiescent conditions and active in response to oxidants/electrophiles.

We recently used positional cloning to identify the transcription factor Nrf2 (NF-E2 related factor 2) as a susceptibility gene in a murine model of oxidant-induced acute lung injury (ALI). NRF2 binds to antioxidant response elements (ARE) and up-regulates protective detoxifying enzymes in response to oxidative stress. This led us to investigate NRF2 as a candidate susceptibility gene for risk of development of ALI in humans. We identified multiple single nucleotide polymorphisms (SNPs) by resequencing NRF2 in ethnically diverse subjects, and one (-617 C/A) significantly (P<0.001) diminished luciferase activity of promoter constructs containing the SNP and significantly decreased the binding affinity (P<0.001) relative to the wild type at this locus (-617 CC). In a nested case-control study, patients with the -617 A SNP had a significantly higher risk for developing ALI after major trauma (OR 6.44; 95% CI 1.34, 30.8; P=0.021) relative to patients with the wild type (-617 CC). This translational investigation provides novel insight into the molecular mechanisms of susceptibility to ALI and may help to identify patients who are predisposed to develop ALI under at risk conditions, such as trauma and sepsis. Furthermore, these findings may have important implications in other oxidative stress related illnesses.

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The effect of cyclophosphamide (CY) on ovarian function was studied in patients with breast cancer receiving prolonged daily administration of this agent (100 mg/day) after radical surgery. Out of 18 premenopausal patients that received 8.4-39.9 g CY, 15 developed permanent amenorrhea. The average dose given before the onset of amenorrhea was 5.2 g in patients in their 40s and 9.3 in their 30s. Urinary estrogens and serum progesterone were measured weekly for approximately 6 months postoperatively in six patients receiving CY. After the onset of amenorrhea, the levels of both hormones ceased to show their normal cyclic changes and remained low persistently, meanwhile serum FSH and LH were markedly elevated. No ovarian follicle was histologically found in three amenorrheic patients who underwent therapeutic oophorectomy after CY therapy. These findings indicate that CY induced primary ovarian failure.

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Specific interactions between transcription factors and cis -acting DNA sequence motifs are primary events for the transcriptional regulation. Many regulatory elements appear to diverge from the most optimal recognition sequences. To evaluate affinities of a transcription factor to various suboptimal sequences, we have developed a new detection method based on the surface plasmon resonance (SPR) imaging technique. Transcription factor MafG and its recognition sequence MARE ( Ma f r ecognition e lements) were adopted to evaluate the new method. We modified DNA immobilization procedure on to the gold chip, so that a double-stranded DNA array was successfully fabricated. We further found that a hydrophilic flexible spacer composed of the poly (ethylene glycol) moiety between DNA and alkanethiol self-assembled monolayers on the surface is effective for preventing nonspecific adsorption and facilitating specific binding of MafG. Multiple interaction profiles between MafG and six of MARE-related sequences were observed by the SPR imaging technique. The kinetic values obtained by SPR imaging showed very good correlation with those obtained from electrophoretic gel mobility shift assays, although absolute values were deviated from each other. These results demonstrate that the double-stranded DNA array fabricated with the modified multistep procedure can be applied for the comprehensive analysis of the transcription factor-DNA interaction.