Yoshihisa Sudo

Development and application of a stable HeLa cell line capable of site-specific transgenesis using the Cre-lox system : Establishment and application of a stable TNFRI knockdown cell line to cytotoxicity assay

Mammalian cell models for gene knock-out/knock-in experiments are important for functional analysis of genes and have a potential of useful tool for toxicological studies. However, uncontrolled insertion of transgenes has raised significant concerns over unwanted side effects. To address this issue, we established a stable HeLa55 cell line capable of site-specific transgenesis by means of Cre-mediated cassette exchange at a site on the long arm of human chromosome 9 containing no constitutive transcripts. We applied HeLa55 to transgenesis of the green fluorescent protein (GFP) gene based on recombinase-mediated cassette exchange. The transformants stably expressed GFP transgenes, even after cryopreservation, without compromising physiological properties. We produced an RNA interference (RNAi)-inducible knockdown stable cell line against human tumor necrosis factor (TNF) receptor I, and one cloned stable cell line (TNFRIKD cells) exhibited long-term gene silencing with significant reduction (ca. 85%) and markedly resisted cytotoxicity induced by TNFalpha. Furthermore, xenobiotics were exposed to stable TNFRIKD cells and different cytotoxicity was exhibited based on various toxicological properties. Thus, we showed the feasibility of RNAi-based stable knockdown cells for xenobiotics-induced cytotoxicity, and HeLa55 has wide application for the generation of stable knock-in and knock-down cells mediated by RNAi.

Unique bioconcentration characteristics of new aryl fluoroalkyl ethers in common carp (Cyprinus carpio)

The bioconcentration factors (BCFs) of seven new aryl fluoroalkyl ethers--four bis-4-tetrafluoroethoxyphenyl-type (bis-type) compounds and three mono-4-tetrafluoroethoxyphenyl-type (mono-type) compounds--were obtained by bioconcentration tests using common carp. The BCFs of 4 of the 7 ethers were higher than 5000, indicating their high bioconcentration potential. The bioconcentration characteristics of the bis-type compounds were different from those of the mono-type compounds and non-fluoro diphenylmethanes with a similar skeleton structure to the bis-type compounds, in taking longer to reach a plateau and having a slower elimination rate and in their distribution patterns in the fish body. The BCF of 1 bis-type compound was much higher than the value predicted by an accepted correlation equation between BCF and P(ow). In addition, the logP(ow) of the bis-type compounds calculated by commercially available computer software was remarkably different from that measured.

Method for the preparation of restricted access media by low-temperature plasma treatment.

A new method for the preparation of restricted access media (RAM) was developed. A low-temperature plasma treatment removed octadecyl groups on the external surface of octadecylsilylated silica gel before those on the internal surface to produce silanol groups. The silanol groups produced were glycerylpropylsilylated to give RAM, DIOL-ODSs, consisting of internal octadecyl groups and external glycerylpropyl groups. DIOL-ODSs were inert to serum proteins and retained low-molecular-mass analytes adequately. Direct injection analysis of anticonvulsants in serum was accurately performed by column-switching high-performance liquid chromatography using a pre-column packed with DIOL-ODS.

Chemical modification of silica-based packing materials for high-performance liquid chromatography

Silica gel is the most widely used as a packing material for HPLC.It possesses the advantages of high mechanical strength, a large number of theoretical plates, ease of chemical modification of the surface and availability at a relatively low price.The demand for further improvements in the performance of silica-based packing materials has been increasing.In this study, improvements of reversedphase packing materials, restricted access media(RAM)and chiral stationary phases were achieved through the development of new methods for silica surface modification.An end-capping method using high-temperature silylation was developed, which produced octadecylsilylated silica gel(ODS)without and undesirable effects of residual silanol groups.In a study on RAM, glycerylalkylsilylated silica gels with alkyl moiety ranging from C3 to C6 were prepared as a new type of RAM.These packing materials did not adsorb proteins, but could retain small molecules, such as drugs, in direct injection analysis of serum samples.This is one of the simplest methods for preparing RAM.In the addition, a low-temperature plasma treatment was applied to the preparation of RAM.The plasma more easily removed octadecyl groups on the external surface of ODS to produce silanol groups than those on the internal surface.The silanol groups were glycerylpropylsilylated to produce RAM.Furthermore, some novel chiral stationary phases were prepared by covalently bonding chiral binaphthol and its derivatives to silica gels.These stationary phases showed enantioselectivity mainly for basic compounds.The hydroxyl groups of binaphthol played a dominant role in chiral recognition and retention.