BUNSEKI KAGAKU Abstracts

The quantitative evaluation of ‘invisible’ radiation exposure is essential to predict health effects and to provide appropriate medical care and evaluation concerning the prognosis of exposed persons. Because it is usually difficult to carry out physical dose measurements by dosimeters in the case of unexpected radiation exposure during accidents or disasters, post hoc dose evaluations using biological specimen of exposed people, called “biological dosimetry”, is indispensable. Although many dose evaluation methods have been developed up to now, the cytogenetic method used to measure the chromosomal aberration frequency of lymphocytes is frequently used as the gold standard from the perspective of accuracy. In addition to that, in this manuscript several applicable dose evaluation methods using biological specimens, such as red blood cells, teeth, nucleic acids and small metabolites, are described and discussed.

Temperature is a fundamental physical quantity that governs every biological reaction within living cells, and the temperature distribution reflects the cellular thermodynamics and function. In medical studies, the cellular pathogenesis of diseases (e.g., cancer) is characterized by extraordinary heat production. Therefore, intracellular temperature imaging of living cells should promote a better understanding of cellular events and the establishment of novel diagnoses and therapies. However, intracellular temperature measurements in living cells have not yet been performed because no thermometry has been available. Here, we present our novel methods for intracellular temperature imaging based on a fluorescent polymeric thermometer and their applications to the monitoring and mapping of the intracellular temperature. We have demonstrated the first intracellular thermometry with a fluorescent nanogel thermometer. The fluorescence response of the thermometer with increasing temperature was independent of the KCl concentration, the environmental pH, or surrounding proteins. Fluorescence imaging of the thermometer in single COS7 cells showed the temperature-dependent response upon heating, which provides the calibration curve for intracellular thermometry. Next, we developed a novel fluorescent polymeric thermometer that could diffuse throughout the cell, and applied it to intracellular temperature mapping, where the fluorescence lifetime of the thermometer was adopted as a temperature-dependent variable. Observations of the fluorescence lifetime of the thermometer in a living cell using fluorescence lifetime imaging microscopy (FLIM) allowed intracellular temperature imaging. The intracellular temperature distribution that we observed indicated that the nucleus and centrosome of a COS7 cell both showed a significantly higher temperature than the cytoplasm, and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. Finally, intracellular temperature variations induced by FCCP (mitochondria uncoupler) were investigated. Both temperature monitoring and imaging showed that the uncoupling of mitochondria provoked the local temperature increase, suggesting that mitochondria undertakes thermogenesis in living cells. Our findings about intracellular temperature demonstrate an intrinsic connection between the temperature and cell function. Thus, our intracellular temperature imaging has a significant impact on the comprehension of cell function, and will provide insights into the regulatory mechanisms of intracellular signaling.

There are many redox reactions to maintain the normal physiological conditions in the living body. A breakdown of the redox balance such as an overproduction of reactive oxygen/nitrogen species and a decrease antioxidant defense may lead to oxidative stress. It can also induce many pathological conditions such as neurological disorders, inflammation, cancer, and ageing. Imaging of the tissue redox status could have one of the markers for drug discovery and clinical applications for novel diagnosis. Nitroxyl radicals have been utilized as a redox sensitive contrast agent in magnetic resonance imaging (MRI) and dynamic nuclear polarization (DNP)-MRI. Recently, we developed home-made DNP-MRI using the circularly transporting system of a sample for high-sensitive and redox analysis based on the anatomical structure by MRI. In this report, we demonstrate the application of the nitroxyl radical to magnetic resonance technique for monitoring the tissue redox status non-invasively.

We have developed a direct mass-spectrometry method that enables speedy molecular analysis of a live single cell under microscopic observation, called “Live Single-cell Mass Spectrometry”. This method can micro-suck subcellular components of a cell, which makes possible direct and real-time metabolic analyses of the cell behavior-linked molecular profile. Furthermore, by using stable isotope-labeled reagents we can trace certain metabolic processes in a live single cell. In this study, we applied this method to analyze the localizations of the histidine metabolites, and to trace their metabolic pathways in the organelle of a rat basophilic leukemia (RBL-2H3) cells.

Basic principles for characterizing molecular adsorbates on a flat surface using infrared and Raman spectroscopy are described. The theoretical base for analyzing the infrared spectra of a condensed matter is electrodynamics. The conclusions of the theoretical analyses depending on an optical configuration are called surface selection rules, which are conveniently used for discussing the molecular orientation on the surface. A theoretical treatment of Raman spectra is also described briefly.

Liquid chromatography/quadrupole time-of flight-mass spectrometry (LC/TOFMS), which combined a new data minding technique and multivariate analysis, enabled the visualization of LC/MS data. Target screening and non-target screening methods for the multi residue analysis of pesticides in food stuff by LC/TOFMS using a new data minding technique, such as Molecular Feature Extraction (MFE) were described. Furthermore, multivariate analysis and a data minding technique, such as MFE for the identification of the unique compound in food stuff and an adequate classification of crops were useful techniques.

This paper describes a high-performance visual threshold detection system for Fe^III at the ppb level. The technique is based on rapid changes of the mole fraction of ML- and M₂L-type complex species about a ligand in a narrow range of Fe^III concentration in a small excess, in case the second metal ion is bound to methylthymol blue (MTB), which can bind two metal ions at both ends of a π-electron conjugated system. The boundary of the color region in the Fe^III concentration is readily controlled by the concentration of MTB. In this study, the visual threshold detection system was combined with the concentration technique using finely ground anion-exchange resin particles to improve the sensitivity. The complex species in a slightly acid solution were adsorbed quantitatively to the finely ground anion-exchanger. Filtration of the ion-exchanger using a membrane filter made of cellulose acetate brought about a superior color property with bathochromic shifts, as well as an enrichment effect. A color transition between the complementary colors through colorlessness, which was caused by the mixing of metanyl yellow as an inert dye, engendered successful performance of the visual threshold detection of Fe^III at the ppb level.

In recent years, we have produced next-generation highly sensitive test papers named ‘dye nanoparticle-coated test strips (DNTSs)’ for harmful ions at ppb level in aqueous samples. The preparative method based on re-precipitation method is simple and feasible, but applicable indicator dyes are limited to be hydrophobic one. In order to fabricate DNTSs with hydrophilic charged indicators, we here report a novel method to prepare DNTSs loaded nano-composites of anionic dyes and ion exchange nano-adsorbents. We tested four popular anionic reagents (tetra-sulfonated TPPS; tri-sulfonated HNB; di-sulfonated Bathophen-S; mono-sulfonated Lumogallion) and two ion exchange nano-adsorbents [weakly basic alumina fiber, AS3 (10 nm×100 nm) and strongly basic trimethyl-modified latex, latex-N(CH₃)₃⁺ (100 nm in diameter)]. The preparative method of nano-composite coated DNTSs is also simple; initially an anionic indicator and a nano-adsorbent are mixed in pH-controlled water, and then the dispersion is filtered with a membrane filter having a 0.1 μm pore size under suction. The retained reagent percentage on the membrane strongly depends on ion exchange nano-adsorbent, the charge of an anionic dye, and the reagent concentration, because the nano-composite formation is based on electrostatic interaction. Especially, by using latex-N(CH₃)₃⁺, almost 100% coating of anionic dyes was successful in the pH range of 2–10. Particle size analysis and zeta potential measurements indicated that nano-composite layers are made in two ways: one is surface filtration of the reagent-adsorbed ion-exchanger; the other is filtration of aggregates of reagent-adsorbed ion-exchangers. The thickness of the nano-composites layer depended on the surface concentration of nano-adsorbent as well as the zeta potential of the nano-composite. The thicknesses of nano-composite layers were 4.55 μm for TPPS/latex-N(CH₃)₃⁺ and 3.34 μm for TPPS/AS3 when the surface concentrations of nano-adsorbents were adjusted to be 0.26 mg cm⁻² and 0.32 mg cm⁻², respectively. Highly negatively charged dyes, like TPPS and HNB, are applicable to the detection of metal ion by filtration enrichment of a sample solution containing high concentration of NaCl; however, mono-anionic or di-anionic dyes, such as Lumogallion and Bathophen-S, are inapplicable due to the leakage of dye or signaling complexes with target ions.

We performed a study of MALDI-MS imaging that is a combination of an optical microscopy and MALDI mass spectroscopy. Mapping images of chemical materials were successfully obtained by applying 2,5-dihydroxybenzoicacid (DHB) as a matrix with a sublimation method. In this paper, we present two results: (1) the distribution of light stabilizers in a multi-layer synthetic film, and (2) the state of the penetration phenomena of hair conditioner chemical components in the hair shaft. In either case, a careful selection of m/z ion enabled successful mapping for individual components; and a variation of the distribution according to the type of components was revealed. Further, important implications with regard to the development design of the materials structure and chemical components were deduced. These results indicated that the MALDI-MS imaging method, which has been extensively used only for biomaterials, is also a powerful tool for the analysis of chemical materials.