Recollection of the early years of the research on cytochrome P450

Since the publication of the first paper on "cytochrome P450" in 1962, the biochemical research on this novel hemoprotein expanded rapidly in the 1960s and the 1970s as its principal roles in various important metabolic processes including steroid hormone biosynthesis in the steroidogenic organs and drug metabolism in the liver were elucidated. Establishment of the purification procedures of microsomal and mitochondrial P450s in the middle of the 1970s together with the introduction of molecular biological techniques accelerated the remarkable expansion of the research on P450 in the following years. This review paper summarizes the important developments in the research on P450 in the early years, for about two decades from the beginning, together with my personal recollections.     

Evaluation of ferritin-overexpressing brain in newly developed transgenic mice

Aberrant expression of ferritin, a major iron-binding protein, has shown to be involved in neurodegenerative diseases. In this study, we generated transgenic (Tg) mice of human ferritin heavy chain (FTH) gene and investigated the effects of ferritin overexpression in FTH-Tg brain by ¹H-MRI and ¹H-MRS. The mice displayed no apparent neurological symptoms, and no specific morphological and T₂ alterations were found in the brain by MRI, and not even by histological studies. ¹H-MRS, however, revealed that some metabolic markers were significantly altered in FTH-Tg brains compared to wild-type control brains, such as decreases in myo-inositol and glutamine, and an increase in lactate. Our present studies suggested that despite the absence of neurological, morphological, T₂, and histological signatures, brain metabolisms were significantly affected in FTH-Tg mice. This study also highlights the usefulness of ¹H-MRS in the analysis of transgenic mouse models.     

Synthesis of the sulfonated condensed polynuclear aromatic (S-COPNA) resins as strong protonic acids

The mixture (PXG/PR=1.00) of pyrene (PR) and p-xylylene glycol (1,4-benzenedimethanol) (PXG) in the presence of 5 wt % of p-toluenesulfonic acid (TsOH) was heated at 140 °C for 45 min under nitrogen to give the highly viscous condensed polynuclear aromatic (COPNA) resin. It was converted into an infusible and insoluble solid by further heating at 300 °C for 1 h. The obtained material was treated with fuming sulfuric acid at 80 °C for 15 h under nitrogen to give the sulfonated COPNA resin. The similar acidic resin was prepared by the reaction of phenanthrene or naphthalene with PXG in the presence of TsOH followed by sulfonation. The performance of the sulfonated polymers as the strong protonic acids was evaluated.     

Monitoring the site-specific incorporation of dual fluorophore-quencher base analogues for target DNA detection by an unnatural base pair system

We developed intramolecular dual fluorophore-quencher base analogues for site-specific incorporation into DNA by an unnatural base pair replication system. An unnatural base pair between 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) exhibits high fidelity in PCR amplification, and the 2-nitropyrrole moiety of Px acts as a quencher. Deoxyribonucleoside triphosphates of Px linked with a fluorophore (Cy3, Cy5 or FAM) were chemically synthesized, and the fluorescent properties and the enzymatic incorporation of the fluorophore-linked dPxTPs into DNA were examined in PCR amplification. The fluorophore-linked dPxTPs were site-specifically incorporated by PCR into DNA, opposite Ds in templates, with high selectivity. Furthermore, we found that the fluorescence of the triphosphates was partially quenched, but increased upon their incorporation into DNA. These dual fluorophore-quencher base analogues would be useful for site-specific DNA labeling and for monitoring the amplification products of target nucleic acid molecules with a specific sequence. We have demonstrated the utility of the fluorophore-linked Px substrates and the Ds-Px pairing in real-time quantitative PCR for target DNA molecule detection.     

Dissipative crystallization of sodium salts of poly (d-glutamic acid), poly (l-glutamic acid), and their low molecular weight analogs

Drying dissipative patterns were observed at room temperature on a cover glass, a watch glass, and a Petri glass dish during the course of dryness of aqueous solution of sodium salts of poly (d-glutamic acid), poly (l-glutamic acid), and their low molecular weight analogs, monosodium d-glutamate, monosodium l-glutamate, and monosodium dl-glutamate. The low molecular weight analogs were hygroscopic and their drying patterns were observed in a dry box coexisted with the bags of desiccant. The broad rings, which are the typical macroscopic drying patterns, were observed for all the samples. Optical isomeric effects on the drying patterns were not recognized. Spherulite (or hedrite) and rod-like crystals from the assemblies of helical main chains of the polymers are formed mainly at the inner area from the broad ring (except central area) and the broad ring area, respectively. Coexistence of sodium chloride enhanced the crystal structures by the cooperative interactions between the polymers and the salts. The typical dissipative crystallization such as accumulation, segregation, and orientation effects of crystals were observed in the drying patterns.     

Colloidal crystallization of thermo-sensitive gel spheres of poly (N-isopropyl acrylamide). Influence of degree of cross-linking of the gels

Morphology, phase diagram, and reflection spectroscopy of the colloidal crystals of thermo-sensitive gel spheres, poly (N-isopropylacrylamide) having degrees of cross-linking 10 and 2?mol.% (pNIPAm(200?C10) and pNIPAm(200?C2)) were studied. Giant colloidal single crystals formed at very low gel concentrations. Critical concentrations of melting increased as the degree of cross-linking decreased in the range from 10 to 0.5?mol.% and/or suspension temperature increased from 20 to 45?°C. The critical concentration decreased sharply as the suspensions were deionized with coexistence of the mixtures of cation- and anion-exchange resins. Density of a gel sphere (gel concentration in weight percent divided by that in volume percent) increased sharply as the degree of cross-linking and/or temperature increased. These results demonstrated that the colloidal crystallization takes place by the extended electrical double layers formed around the gel spheres in addition of the excluded-volume effect of the gels. Most of the researchers including the authors have believed that the crystallization of the gel spheres takes place by the excluded-volume effect. However, the present work clarified that the colloidal interfaces, which are inevitable for the formation of the electrical double layers, are formed firmly between the water phase and gel spheres, though the gel spheres contain a lot of water molecules in the sphere region.     

Nitrogen doping into titanium dioxide by the sol–gel method using nitric acid

N-doped TiO₂ has been prepared by use of sol–gel systems containing titanium alkoxide, with nitric acid as the nitrogen source. The time needed for gelation of the systems was drastically reduced by ultrasonic irradiation. The peaks assigned to the nitrate and nitrous ions were observed by FT-IR measurement during the sol–gel reaction. The N-doping was confirmed by the observation of N–O peaks in the XPS spectrum of the sample heated at 400 °C. The nitrate ion acted as an oxidizer of the ethanol solvent and titanium species. The TiO₂ became doped with nitrogen oxide species as a result of reduction of nitrate ion incorporated into the dried gel samples. These results indicated that the added nitric acid was reduced during the sol–gel transition and heating process, and the resulting NO species were situated in the titania networks. The UV and visible photocatalytic activity of the samples was confirmed by the degradation of trichloroethylene.     

Dissipative crystallization of aqueous mixtures of potassium salts of poly(riboadenylic acid) and poly(ribouridylic acid)

Dissipative drying patterns of aqueous mixtures of potassium salts of poly(riboadenylic acid) (KPolyA) and poly(ribouridylic acid) (KPolyU) were studied on a cover glass, a watch glass and a glass dish at room temperature. Accumulation of the polymers forming the broad rings near the outside edge and the inner area of the dried film was observed. The fine multiple ring structures formed when the affinity of the polymer with the substrate is strong. Microscopic drying patterns changed drastically depending on the location in the dried film. Microscopic drying patterns were mainly dendritic long rods and sword (halberd)-like rods. They are assigned to the crystals of double-stranded and triple-stranded helices of the A:U and A:2U complexes, respectively. Cross-like drying patterns are also observed originated from the salt-polymer interaction.     

Photocatalytic reaction on photofuel cell titania electrode

Benzoic acid-doped titania electrodes were prepared from titanium alkoxide sols containing benzoic acid in order to examine the photocatalytic reaction of the fuel material concentrated on the titania surface of a photofuel cell electrode. This doping was developed in order to understand the physicochemical processes on the titania rather than to advance the practical use of the photofuel cells. The observed photocurrent and CO₂ and H₂O productions indicated that the oxidation of the benzoic acid enhanced the generation of electricity during the UV irradiation. Benzoic acid molecules should be oxidized by oxygen molecules and holes on the titania surface. The steam treatment of the electrodes improved the benzoic acid oxidation and the photocurrent because it promoted the titania densification and enhanced the interaction between the benzoic acid and titania. The benzoic acid-doped titania is a valid model of the fuel material concentrated in the porous titania when using benzoic acid as the fuel material. The contact between the benzoic acid and titania is important in order to obtain a high photofuel electric conversion.