53 research outputs found
Biochemical Comparison of Anopheles gambiae and Human NADPH P450 Reductases Reveals Different 2′-5′-ADP and FMN Binding Traits
NADPH-cytochrome P450 oxidoreductase (CPR) plays a central role in chemical
detoxification and insecticide resistance in Anopheles gambiae,
the major vector for malaria. Anopheles gambiae CPR (AgCPR) was
initially expressed in Eschericia coli but failed to bind
2′, 5′-ADP Sepharose. To investigate this unusual trait, we
expressed and purified a truncated histidine-tagged version for side-by-side
comparisons with human CPR. Close functional similarities were found with
respect to the steady state kinetics of cytochrome c reduction,
with rates (kcat) of 105
s−1 and 88 s−1, respectively, for mosquito
and human CPR. However, the inhibitory effects of 2′,5′-ADP on
activity were different; the IC50 value of AgCPR for 2′,
5′ –ADP was significantly higher (6–10 fold) than human CPR
(hCPR) in both phosphate and phosphate-free buffer, indicative of a decrease in
affinity for 2′, 5′- ADP. This was confirmed by isothermal titration
calorimetry where binding of 2′,5′-ADP to AgCPR
(Kd = 410±18 nM) was
∼10 fold weaker than human CPR
(Kd = 38 nM). Characterisation
of the individual AgFMN binding domain revealed much weaker binding of FMN
(Kd = 83±2.0 nM) than the equivalent
human domain (Kd = 23±0.9 nM).
Furthermore, AgCPR was an order of magnitude more sensitive than hCPR to the
reductase inhibitor diphenyliodonium chloride
(IC50 = 28 µM±2 and 361±31
µM respectively). Taken together, these results reveal unusual biochemical
differences between mosquito CPR and the human form in the binding of small
molecules that may aid the development of ‘smart’ insecticides and
synergists that selectively target mosquito CPR
The flavonoid galangin is an inhibitor of CYP1A1 activity and an agonist/antagonist of the aryl hydrocarbon receptor
The effect of the dietary flavonoid galangin on the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA), the activity of cytochrome P 450 1A1 (CYP1A1), and the expression of CYP1A1 in MCF-7 human breast carcinoma cells was investigated. Galangin inhibited the catabolic breakdown of DMBA, as measured by thin-layer chromatography, in a dose-dependent manner. Galangin also inhibited the formation of DMBA-DNA adducts, and prevented DMBA-induced inhibition of cell growth. Galangin caused a potent, dose-dependent inhibition of CYP1A1 activity, as measured by ethoxyresorufin-O-deethylase activity, in intact cells and in microsomes isolated from DMBA-treated cells. Analysis of the inhibition kinetics by double-reciprocal plot demonstrated that galangin inhibited CYP1A1 activity in a non-competitive manner. Galangin caused an increase in the level of CYP1A1 mRNA, indicating that it may be an agonist of the aryl hydrocarbon receptor, but it inhibited the induction of CYP1A1 mRNA by DMBA or by 2,3,5,7-tetrachlorodibenzo-p-dioxin (TCDD). Galangin also inhibited the DMBA- or TCDD-induced transcription of a reporter vector containing the CYP1A1 promoter. Thus, galangin is a potent inhibitor of DMBA metabolism and an agonist/antagonist of the AhR, and may prove to be an effective chemopreventive agent. © 1999 Cancer Research Campaig
SEISMIC WIDE ANGLE MEASUREMENTS IN THE BAVARIAN MOLASSE BASIN*. THE GERMAN RESEARCH GROUP FOR EXPLOSION SEISMOLOGY
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