ABSTRACT
Tobacco cytochrome P450 (CYP) 71AH11 metabolized the herbicide chlorotoluron, and its mRNA level was increased in tobacco culture cells by the treatment of 2,4-D. In order to clarify molecular mechanisms of induced gene expression of CYP71AH11 by herbicide treatment, a 1574-bp 5'-flanking region of CYP71AH11 was cloned, ligated to the reporter ß-glucuronidase (GUS) gene, and then transformed into tobacco plants. The GUS activity in the transgenic tobacco plants was highly induced by bromoxynil treatment, followed by 2,4-D. Chlorotoluron was slightly increased the GUS activity. The bromoxynil-increased GUS activity was partially repressed by the antioxidants, suggesting that reactive oxygen species may be involved in activation of the 5'-flanking region of CYP71AH11 by bromoxynil treatment. Deletion and mutation assays showed that the region CD (-1281 to -770bp from the start codon of CYP71AH11) was important, but not sufficient, for response to bromoxynil. Electrophoretic mobility shift assays and southwestern blotting revealed that the sequences AAAAAG, and GAACAAAC and GAAAATTC in the CD region were important for interaction to the nuclear proteins of <30 and ≈75 kDa, respectively. Particularly, interaction between AAAAAG and <30 kDa proteins was increased by bromoxynil treatment. These results gave a cue for understanding the bromoxynil-induced gene expression of CYP71AH11, which may contribute to herbicide tolerance and selectivity in crop plants.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Gene Expression Regulation, Enzymologic , Herbicides/metabolism , Nicotiana/enzymology , Phenylurea Compounds/metabolism , Plant Proteins/metabolism , Base Sequence , Cytochrome P-450 Enzyme System/genetics , Gene Expression Regulation, Plant , Molecular Sequence Data , Plant Proteins/genetics , Promoter Regions, Genetic , Nicotiana/genetics , Nicotiana/metabolismABSTRACT
PURPOSE: The chemotherapeutic effects and pharmacokinetics properties of WP-0405 (a thermo-setting in situ 0.3% ofloxacin-containing ophthalmic gel) and ofloxacin (a conventional 0.3% ofloxacin solution) on methicillin-resistant Staphylococcus aureus (MRSA) keratitis were compared in a rabbit model. METHOD: The single-instillation pharmacokinetics of WP-0405 and ofloxacin in the cornea, aqueous humor, conjunctiva, and iris-ciliary body were determined in normal rabbit eyes. To compare the duration of antimicrobial action, WP-0405 or ofloxacin was instilled oncedaily in an early-treatment model of keratitis, and corneas were either removed immediately or 4 or 8 h postinstillation. In another experiment, WP-0405 was instilled two or three times daily to compare its antibiotic efficacy with three-times daily instillation of ofloxacin in the same early-treatment model of keratitis; corneas were then removed after determining the extent of the abscess area. In another experiment, WP-0405 was instilled four or eight times daily to compare its effects with eight-times daily instillation of ofloxacin in a late-treatment model of keratitis, and corneas were removed. The number of viable bacteria in the corneas was determined in all experiments. RESULTS: Cmax and AUC0- in tissues treated with WP-0405 were 1.5-3.4-fold and 1.8-2.9-fold greater than those treated with ofloxacin, respectively. WP-0405 significantly reduced the number of viable bacteria for up to 8 h after a single instillation. WP-0405 not only significantly reduced the number of viable bacteria, but also the size of the abscess area at the same frequency of instillation. When compared to ofloxacin, WP-0405 exhibited an approximately equivalent antibiotic effect, with fewer administrations. CONCLUSIONS: As a result of its pharmacokinetics, WP-0405 had a more potent, longer-acting antibiotic effect than did ofloxacin. Furthermore, because of its lower required instillation frequency, which would improve patient compliance, WP-0405 has great potential therapeutic benefits.
