Amphipol-facilitated elucidation of the functional tetrameric complex of full-length cytochrome P450 CYP2B4 and NADPH-cytochrome P450 oxidoreductase.

Interactions of membrane-bound mammalian cytochromes P450 (CYPs) with NADPH-cytochrome P450 oxidoreductase (POR), which are required for metabolism of xenobiotics, are facilitated by membrane lipids. A variety of membrane mimetics, such as phospholipid liposomes and nanodiscs, have been used to simulate the membrane to form catalytically active CYP:POR complexes. However, the exact mechanism(s) of these interactions are unclear because of the absence of structural information of full-length mammalian CYP:POR complexes in membranes. Herein, we report the use of amphipols (APols) to form a fully functional, soluble, homogeneous preparation of full-length CYP:POR complexes amenable to biochemical and structural study. Incorporation of CYP2B4 and POR into APols resulted in a CYP2B4:POR complex with a stoichiometry of 1:1, which was fully functional in demethylating benzphetamine at a turnover rate of 37.7 ± 2.2 min-1, with a coupling efficiency of 40%. Interestingly, the stable complex had a molecular weight (Mw) of 338 ± 22 kDa determined by multiangle light scattering, suggestive of a tetrameric complex of 2CYP2B4:2POR embedded in one APol nanoparticle. Moreover, negative stain electron microscopy (EM) validated the homogeneity of the complex and allowed us to generate a three-dimensional EM map and model consistent with the tetramer observed in solution. This first report of the full-length mammalian CYP:POR complex by transmission EM not only reveals the architecture that facilitates electron transfer but also highlights a potential use of APols in biochemical and structural studies of functional CYP complexes with redox partners.

Comparative study of polymer containing beta-cyclodextrin and -COOH for adsorption toward aniline, 1-naphthylamine and methylene blue.

Three different polymers P1, P2 and P3 (P1 containing both beta-CD and -COOH, P2 containing beta-CD and P3 containing -COOH) were synthesized and applied to adsorption toward aniline, 1-naphthylamine and methylene blue. The concentrations (C) before and after adsorption were determined and the adsorption capacities (q) of P1, P2 and P3 were calculated. The maximum adsorption capacities (q(max)) toward aniline: q(max) (P1)=104 micromol g(-1), q(max) (P2)=14.9 micromol g(-1) and q(max) (P3)=53.1 micromol g(-1); toward 1-naphthylamine: q(max) (P1)=184 micromol g(-1), q(max) (P2)=53.8 micromol g(-1) and q(max) (P3)=125 micromol g(-1); toward methylene blue: q(max) (P1)=200 micromol g(-1), q(max) (P2)=12.7 micromol g(-1) and q(max) (P3)=215 micromol g(-1). P1 exhibited remarkable adsorption toward all the three adsorbates. P2 was almost equal to P1 in adsorption toward methylene blue, but was less efficient than P1 in adsorption toward aniline and 1-naphthylamine. P3 also exhibited considerable adsorption toward aniline and 1-naphthylamine, but was inefficient toward methylene blue. P1 was obtained from nontoxic materials and through environment friendly procedures, so it was potentially an efficient and green adsorbent for water purification.

Enzymatic responses of human deciduous pulpal fibroblasts to dental restorative materials.

The purpose of this study was to evaluate the responses of succinic dehydrogenase (SDH) and alkaline phosphatase (ALP) activities of human deciduous teeth pulpal fibroblasts (HDPF) to dental restorative materials. Tested materials included Z100 (3M), Dyract (Dentsply), FujiII (GC), and FujiIILC (GC). IRM (Dentsply) and culture medium (MD) alone were used as positive and negative controls, respectively. Specimens 6 mm (diameter) x 3 mm were prepared in accordance with manufacturers' instructions. For light-cured materials, specimens were light cured for 40 s on both sides under a celluloid strip. For chemical-cured materials, specimens were allowed to set at room temperature for 15 min. The specimens were immersed in 1 mL of culture medium without serum for 24 h at room temperature. The extracts were filtered through 0.22-mm filters. HDPF (10,000 cells/well) was incubated with 100 microL of extract and 20 % FBS in a 96-well plate for 24 h in a 37 degrees, 5 % CO(2) incubator. Six wells per material were prepared. Optical density (OD) of SDH and ALP of HDPF were measured by a spectrophotometer. The means were analyzed by ANOVA and then a Duncan Test. The ranking of OD of SDH was IRM < FujiIILC < FujiII = Z100 < Dyract < MD (p < 0.05). The ranking of OD of ALP was IRM < Z100 = Dyract < FujiII < FujiIILC < MD (p < 0.05). The result showed that all of the tested restorative materials were cytotoxic to human deciduous pulpal fibroblasts. The cytotoxicity of resin-modified glass ionomer cements (FujiIILC) was stronger than that of traditional glass ionomer cements (FujiII) and composite resin (Z100), and that of compomer (Dyract) was the weakest. On the contrary, ALP activities of resin-modified glass ionomer cements (FujiIILC) and composite resin (Z100) were higher than those of traditional glass ionomer cements (FujiII), while those of compomer (Dyract) were the lowest. It is concluded that, in this study, FujiIILC was the most cytotoxic material and the least inhibitive of ALP activities, Dyract was the weakest cytotoxic material and had the highest inhibition of ALP activities. The rankings of the MTT assay and the ALP assay were not consistent.