Enhanced activity of 3alpha-hydroxysteroid dehydrogenase by addition of the co-solvent 1-butyl-3-methylimidazolium (L)-lactate in aqueous phase of biphasic systems for reductive production of steroids.

The enzyme activity of 3alpha-hydrosteroid dehydrogenase (HSDH) was enhanced by the addition of the co-solvent 1-butyl-3-methylimidazolium (L)-lactate ([Bmim][lactate]) to 50 mM Tris-HCl buffer. When utilizing [Bmim][lactate], the reaction velocity of HSDH increased. Also, reductive production of androsterone was investigated in an aqueous-organic solvent biphasic system containing 5% [Bmim][lactate] as the co-solvent of aqueous phase. In a coupled-enzyme system comprising HSDH and formate dehydrogenase (FDH), a two-fold increase in production rate of androsterone was obtained when utilizing [Bmim][lactate] with NADH regeneration.

Enhanced accumulation of anthocyanin in cultured strawberry cells by repetitive feeding of L-phenylalanine into the medium.

L-Phenylalanine (Phe), biosynthetic precursor of anthocyanin, was repetitively added into a suspension culture of strawberry cells, constantly producing anthocyanins. In the repetitive feeding culture, the maximum anthocyanin accumulation per culture was 30% and 81% higher than those in a single Phe-feeding culture and non-feeding culture, respectively. This means that the inhibitory effect of Phe on cell growth was successfully avoided and its intracellular free-state concentration was maintained at a higher level by the repetitive feeding of L-phenylalanine at relatively lower intra-medium concentration. These results support the phenylalanine pool hypothesis which states that the intracellular L-phenylalanine level determines anthocyanin production. However, it was also suggested that there exist other restrictions controlling intracellular anthocyanin accumulation.

Lanthanoid element recognition on surface-imprinted polymers containing dioleylphosphoric acid as a functional host.

Surface-imprinted polymers have been newly developed for the separation of lanthanoid elements: i.e. La(III), Ce(III), and Dy(III). The imprinted polymers were prepared by surface template polymerization with dioleylphosphoric acid, which exhibits a high affinity to lanthanoids, as a functional host molecule. Separation behavior of La(III), Ce(III) and Dy(III) was investigated with the imprinted polymers, and the imprinting effect of the polymers was evaluated in comparison with that of the unimprinted polymers and also with a conventional solvent extraction method for the same lanthanoid ions. The results indicate that the increase of selectivity for Dy(III) compared to the rest of the ions by the surface-imprinted polymers originated from a synergistic effect of both the affinity with the functional host molecule in nature and the size exclusion by the cavity formed on the polymer surface.

Activation of lignin peroxidase in organic media by reversed micelles.

Activation of lignin peroxidase (LIP) in an organic solvent by reversed micelles was investigated. Bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT) was used as a surfactant to form a reversed micelle. Lyophilized LIP from an optimized aqueous solution exhibited no enzymatic activity in any organic solvents examined in this study; however, LIP was catalytically active by being entrapped in the AOT reversed micellar solution. LIP activity in the reversed micelle was enhanced by optimizing either the preparation or the operation conditions, such as water content and pH in water pools of the reversed micelle and the reaction temperature. Stable activity was obtained in isooctane because of the stability of the reversed micelle. The optimal pH was 5 in the reversed micellar system, which shifted from pH 3 in the aqueous solution. The degradation reaction of several environmental pollutants was attempted using LIP hosted in the AOT reversed micelle. Degradation achieved after a 1-h reaction reached 81%, 50%, and 22% for p-nonylphenol, bisphenol A, and 2,4-dichlorophenol, respectively. This is the first report on the utilization of LIP in organic media.

Inhibitory effects of extracts from peels of Citrus natsudaidai encapsulated in hybrid liposomes on the growth of tumor cells in vitro.

Inhibitory effects of extracts from peels of Citrus natsudaidai (natsumikan) encapsulated in hybrid liposomes (HL) composed of L-alpha-dimyristoylphosphatidylcholine and polyoxyethylene (20) sorbitan monolaurate on the growth of tumor cells were examined. The extracts with lower polar solvents inhibited the growth of B-16 mouse melanoma and human lung carcinoma cells, although the extracts with higher polar solvents showed no antitumor activity. In particular, the inhibitory effects of extracts with lower polar solvents encapsulated in HL were enhanced as compared with those of free extracts. Fluorescence microscopic analysis indicated that the HL including petroleum ether extracts induced apoptosis in B-16 mouse melanoma cells. On the other hand, the viability of normal human fibroblast cells was even less affected by the extracts of natsumikan. These results suggest that hydrophobic antitumor agents should be present in peels of natsumikan.

Complex formation of cytochrome C with a calixarene carboxylic acid derivative: a novel solubilization method for biomolecules in organic media.

A calixarene carboxylic acid derivative has been found to form a complex with the cationic protein cytochrome c. The solubilized cytochrome c was stable and showed peroxidase activity in chloroform. The calix[6]arene and the calix[8]arene achieved quantitative extraction of the protein. The calix[6]arene, whose cavity is well-fitted to a protonated amino group, exhibited a selectivity to lysine-rich proteins due to the recognition of the epsilon-amino groups in lysine residues on the surface of the protein. This is the first report showing protein extraction by calixarenes. The solubilized cytochrome c could catalyze an oxidative reaction in organic solvents. This host compound functions as a novel solubilization tool for biomolecules and a separation tool for lysine-rich proteins.

Remarkably enhanced inhibitory effects of three-component hybrid liposomes including sugar surfactants on the growth of lung carcinoma cells.

Sugar parts play important roles in recognizing molecules on the cell membranes. We successfully produced sugar-type micellar surfactants, lactonoalkylamide (LacCn), for the first time. Spherical vesicles, three-component hybrid liposomes, were obtained after the sonication of the mixture of L-alpha-dimyristoyl-phosphatidylcholine (DMPC), Tween 20 and LacCn (DMPC:Tween 20:LacCn=65:7:28). It is noteworthy that high inhibitory effects of the three-component hybrid liposomes composed of DMPC, Tween 20, and LacCn (DMPC:Tween 20:LacCn=65:7:28) on the growth of glioma (U251) and lung adenocarcinoma (RERF-LC-OK) cells were attained in vitro without any drug, although no significant inhibitory effects of any individual component (DMPC, Tween 20, LacCn) or the two-component hybrid liposomes of DMPC and Tween 20 on the growth of tumor cells examined were obtained.

Formation and Characterization of Reversed Micelles Composed of Phospholipids and Fatty Acids.

The formation of reversed micellar systems composed of phosphatidylcholine (PC) and fatty acid was newly demonstrated by a significant increase in water content in the organic ethyl oleate phase when the micelles were prepared by the contact method. The solubilized water concentration in the reversed micellar organic phase reached 3 wt%. The new systems are expected to be used as highly biocompatible reversed micellar systems. The structure of the reversed micelles composed of PC and oleic acid was characterized by determining the water concentration and by small-angle X-ray scattering analysis. The reversed micelles composed of PC and oleic acid formed in ethyl oleate were spherical. The radius of gyration was between 30 and 50 Å. The size of the reversed micelles decreased with an increase in the oleic acid concentration and was independent of the PC concentration. Experimental results indicated that the structure of the reversed micellar system was determined by the oleic acid concentration. An increase in the PC concentration caused an increase in the number of reversed micelles of the same size. These reversed micellar systems are expected to be used as solubilization media in pharmaceutical and food industries because they are not toxic. Copyright 2001 Academic Press.