Izumi Morita

Chemopreventive effects of emodin and cassiamin B in mouse skin carcinogenesis

In continuation of our works of natural and synthetic products as cancer chemopreventive agents, we have examined emodin and cassiamin B, which were isolated from Cassia siamea. These compounds exhibited the remarkable anti-tumor promoting effect on two-stage carcinogenesis test of mouse skin tumors induced by 7,12-dimethylbenz[a]anthracene as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter by both topical application. Furthermore, emodin exhibited potent inhibitory activity on two-stage carcinogenesis test of mouse skin tumors induced by nitric oxide donor, (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide as an initiator and TPA as a promoter.

Inhibitory effects of anthraquinones and bianthraquinones on Epstein-Barr virus activation

Short-term in vitro assays for anti-tumor promoters were carried out for several anthraquinones and bianthraquinones, which were isolated from Cassia siamea and derived from cascaroside A. Anthraquinone monomers showed higher anti-tumor promoting activity than that of bianthraquinones.

Correlation between oxidation potentials and inhibitory effects on Epstein-Barr virus activation of flavonoids.

The oxidation potentials of fifteen flavonoids in phosphate buffer at pH 7.2 were determined by cyclic voltammetry. A good correlation was found between these oxidation potentials and the ability of flavonoids to inhibit Epstein-Barr virus early antigen (EBV-EA) activation. Furthermore, multiple regression analysis revealed that the solvent-accessible surface area (SASA) was a useful parameter for estimating the inhibitory effects of flavonoids on EBV-EA activation.

Correlation of redox potentials and inhibitory effects on Epstein-Barr virus activation of 2-azaanthraquinones

As a continuation of our studies using natural and synthetic products as cancer chemopreventive agents, we examined the standard redox potentials of some 2-azaanthraquinones in phosphate buffer at pH 7.2 by means of cyclic voltammetry. A definite correlation has been found between the redox potentials and the inhibitory effects of the 2-azaanthraquinones on Epstein-Barr virus early antigen (EBV-EA) activation. It has been further shown that the correlation can be enhanced by introducing an electronic properties, i.e. the atomic charges at the C5 and O12 atoms in the quinone skeleton ring and the HOMO energy as additional parameters.

Correlation between Cytotoxic Activities and Reduction Potentials of Heterocyclic Quinones

To search for possible anti-tumor agents or anti-tumor promoters among natural or synthetic products, we used cyclic voltammetry to determine the reduction-oxidation potentials of heterocyclic quinones in phosphate buffer at pH 7.2. We determined the growth inhibitory- and cytotoxic activities of 12 heterocyclic quinone anti-tumor agent candidates against a panel of 39 human cancer cell lines (JFCR39). The average concentrations of the heterocyclic quinones required for 50% growth inhibition (GI50) against JFCR39 ranged from 0.045 to 13.2 μM, and the 50% lethal concentration (LC50) against JFCR39 ranged from 0.398 to 77.7 μM. The average values of GI50 or LC50 of the heterocyclic quinones correlated significantly with their reduction potentials. These results suggested that reduction-oxidation potentials could be a useful method for the discovery of novel antitumor agents.

Correlation between reduction potentials and inhibitions of Epstein–Barr virus activation by anthraquinone derivatives

As a continuation of studies using natural and synthetic products as cancer chemopreventive agents, we used cyclic voltammetry to examine the reduction-oxidation potentials of methylated emodin derivatives prepared from emodin in phosphate buffer at pH 7.2. A good correlation was found between the inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation and the reduction potential of methylated emodin derivatives. Furthermore, there was significant correlation between EBV-EA activation and the reduction potential of 35 anthraquinone derivatives including methylated emodin derivatives. It was further shown that the correlation could be enhanced by including LUMO energy and the number of hydroxy groups as additional parameters.

A monoclonal antibody-based enzyme-linked immunosorbent assay for human urinary cotinine to monitor tobacco smoke exposure

Immunoassays for human urinary cotinine [(S)-(−)-cotinine], a major nicotine metabolite, are useful to monitor the degree of tobacco smoke exposure. However, practical monoclonal antibodies for measuring urinary cotinine are not widely available. Here we generated a monoclonal antibody against a newly prepared cotinine–albumin conjugate and developed an enzyme-linked immunosorbent assay (ELISA). Splenocytes from a BALB/c mouse that had been immunized with the conjugate were fused with P3/NS1/1-Ag4-1 myeloma cells, and a hybridoma clone secreting the anti-cotinine antibody, Ab-CT#45 (IgG1κ; amino acid sequences of the variable domains were determined), was established. This antibody was immobilized on microplates to generate an ELISA system in which biotin-labeled cotinine was used as the tracer molecule. After competitive reactions with analyte, the bound biotin residues were monitored colorimetrically with peroxidase-labeled streptavidin. This ELISA produced dose–response curves for cotinine ranging from 0.40–100 ng per assay and was able to discriminate nicotine, cotinine N-oxide, cotinine N-glucuronide (cross-reactivity was each <0.5%, taking cotinine as 100%), (3′R, 5′S)-3′-hydroxycotinine O-glucuronide (0.6%) and (R, S)-norcotinine (1.5%). Another major nicotine metabolite, (3′R, 5′S)-3′-hydroxycotinine, showed 8.4% cross-reactivity, but an analytical recovery test indicated that this metabolite did not result in a significant overestimation of the urinary cotinine levels. The present ELISA has been validated to allow the urine specimens to be “directly” (without pretreatment) measured in order to screen and monitor tobacco smoke exposure, and if necessary, to enable cotinine alone to be more specifically examined after the urine specimens are subjected to a simple chloroform extraction.

Development of an LC-ESI-MS/MS method for the determination of histamine : Application to the quantitative measurement of histamine degranulation by KU812 cells

A rapid, simple, and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method was developed for the identification and quantification of histamine without a previous derivatization step or the addition of general ion-pairing reagents to the mobile phase. This method was used to measure histamine release following degranulation of KU812 human basophilic cells, using pyrazol as an internal standard. Analyses were performed on an LC system employing a Cosmosil 5C(18) PAQ column and an isocratic elution with methanol-0.005% trifluoroacetic acid (1:1) at a flow rate of 0.2 mL/min. A triple-quadrupole mass spectrometer, equipped with an electrospray ionization interface was employed, operating in the positive ion mode. The retention time of histamine and the internal standard were 4.0 and 5.0 min, respectively. The relative standard deviations (R.S.D.s) of the retention time and peak area were between 0.47% and 2.03%. Micropipette tip solid-phase extraction (SPE) using LooseTip C(18) allowed for not only rapid sample preparation, but also decreased suppression effects, improving peak shape. This method was used to evaluate the anti-allergic effects of compounds contained in Taxus yunnanensis extracts. Four constituents that were isolated from the wood extracts of T. yunnanensis and sodium cromoglicate, which is used as a first line anti-allergic drug, were tested in an in vitro histamine release inhibition assay. Of these compounds, taxiresinol and isotaxiresinol were more inhibitory than sodium cromoglicate.

Gaussia Luciferase as a Genetic Fusion Partner with Antibody Fragments for Sensitive Immunoassay Monitoring of Clinical Biomarkers.

In this study, we show the utility of Gaussia luciferase (GLuc), which is much smaller than previously found luciferases, as the fusion partner with artificial antibody species for developing sensitive immunoassay systems. As an example, we constructed a bioluminescent enzyme-linked immunosorbent assay (BL-ELISA) system determining the major glucocorticoid cortisol. A monoclonal antibody was newly elicited against a cortisol-albumin conjugate, and the genes encoding its variable domains (VH and VL) were cloned and combined to encode a single-chain Fv fragment (scFv). scFv was then linked to the wild-type GLuc gene or that encoding GLuc mutants reported to show improved emission kinetics and expressed in the periplasmic space of several Escherichia coli strains. Notably, the wild-type GLuc fusion protein (scFv-wtGLuc) showed the most suitable luminescent properties for BL-ELISAs. In our system, scFv-wtGLuc was reacted competitively with the analyte and immobilized cortisol moieties, and the bound GLuc activity was monitored with coelenterazine as the substrate. Successful batch-type luminescence detection was achieved using a plate reader without built-in injectors. The midpoint and limit of detection in a typical dose-response curve were 4.1 and 0.26 pg/assay, respectively, thus exhibiting much more sensitivity than conventional cortisol immunoassays. Serum cortisol levels (as the sum with cortisone) for healthy subjects, determined without any pretreatment, were compatible with reported reference ranges. The scFv-wtGLuc probe was stable over a year under storage as periplasmic extracts at -30 °C or with repeated freeze-thawing. These results suggest that GLuc fusions with antibody fragments might serve as useful and highly sensitive immunoassay probes in various clinical settings.

Characterization of emodin metabolites in Raji cells by LC–APCI-MS/MS

A rapid, simple, and sensitive liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS) method was developed for the identification and quantification of emodin metabolites in Raji cells, using aloe-emodin as an internal standard. Analyses were performed on an LC system employing a Cosmosil 5C(18) AR-II column and a stepwise gradient elution with methanol-20mM ammonium formate at a flow rate of 1.0 mL/min operating in the negative ion mode. As a result, the starting material emodin and its five metabolites were detected by analyzing extracts of Raji cells that had been cultivated in the presence of emodin. The identification of the metabolites and elucidation of their structures were performed by comparing their retention times and spectral patterns with those of synthetic samples. In addition to the major metabolite 8-O-methylemodin, four other metabolites were assigned as omega-hydroxyemodin, 3-O-methyl-omega-hydroxyemodin, 3-O-methylemodin (physcion), and chrysophanol.