Bioluminescence color modulation of Vibrio fischeri strain Y1 coupled with alterable levels of endogenous yellow fluorescent protein and its fluorescence imaging

Bioluminescence (BL) (lambda(max) approximately 535 nm) of Vibrio fischeri strain Y1 has been previously characterized in terms of the fluctuation in intracellular levels of yellow fluorescent protein (YFP). In this study fluorescence microscopic analysis has revealed that yellow fluorescence, as well as blue fluorescence attributable to a luciferase intermediate, is localized to the periphery of V. fischeri Y1 cells. This finding indicates that both YFP and the luciferase are present in the vicinity of the cell membrane. By using cyanide to enhance yellow BL, it has been shown that BL modulation is coupled with the fluctuations in the intracellular levels of YFP and the primary emitter. On the basis of the BL characterization, combined with results of a sedimentation experiment, it has been shown that larger cells produce a relatively stronger yellow BL. Two-dimensional gel electrophoresis of cell-protein extracts has shown that the YFP level is more alterable than the luciferase level. It is postulated that the yellow BL modulation takes place in connection with cell growth.     

A further study on the combined use of internal standard and isotope-labeled derivatization reagent for expansion of linear dynamic ranges in liquid chromatography-electrospray mass spectrometry

The combined use of a so-called internal standard and the isotope-labeled derivatization reagent for the quantification of analytes for liquid chromatography-mass spectrometry (LC/MS) was further studied. The sample solution (containing the analytes and an internal standard) was derivatized with the light form of the derivatization reagent, 7-(N,N-dimethylaminosulfonyl)-4-(aminoethyl)piperazino-2,1,3-benzoxadiazole (DBD-PZ-NH(2)) or 7-(N,N-dimethylaminosulfonyl)-4-piperazino-2,1,3-benzoxadiazole (DBD-PZ). A standard solution of the analytes (containing an internal standard) was derivatized with the isotope (d(6))-labeled derivatization reagent, DBD-PZ-NH(2) (D) or DBD-PZ (D), and served as the isotope-labeled internal standards. The peak heights of the targeted analytes derivatives in the sample solution were corrected using those of the internal standard and the heavy form derivatives of the standards, and the calibration curves were constructed. The curve bending of the calibration curves caused by the ion suppression at the ion source was suppressed and the linear dynamic ranges of the calibration curves were expanded. The derivatives of DBD-PZ-NH(2) were about 10 times more sensitively detected than those of DBD-PZ derivatives and, therefore, DBD-PZ-NH(2) might be suitable for sensitive detection.     

LET dependence of the formation of oxidative damage 8-hydroxy-2′-deoxyguanosine (8-OHdG) in 2′-deoxyguanosine aqueous solution irradiated with heavy ions

To evaluate the contribution of indirect action mediated by OH radicals in biological effects of high LET radiations, we examined the production of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in 2′-deoxyguanosine aqueous solution using various ion species with an LET range from 20 to 300 keV/μm. The 8-OHdG yield decreased with increasing LET. In the hypoxic irradiation condition, the yield showed constant or rather increasing tendency above about 100 keV/μm, which is consistent with an oxygen-in-the-track hypothesis to explain the diminishment of oxygen effect.     

Enhanced radiation-induced cell killing by Herbimycin A pre-treatment

Herbimycin A (HA), as in Geldanamycin, binds to conserved pockets of heat shock protein 90 (Hsp90) and inhibits its chaperone functions. Hsp90 plays an integral role in cancer cell growth and survival, because it maintains the stability of several key proteins by its chaperone's activity. It is known that some of the proteins associated with radiation responses are functionally stabilized by Hsp90. In this study, we investigated the effect of HA on radiosensitivity in human cancer cells and the mechanism related to the sensitization. In order to gain a mechanistic insight of this sensitization, we examined repair of DNA double-strand breaks (DSBs) in irradiated human cancer cells pre-treated with HA, as unrepaired DSBs are thought to be the main cause of radiation-induced cell death. Cellular radiosensitivity was determined by clonogenic assay, and the DSB rejoining kinetics was examined by constant field gel electrophoresis. SQ-5, a lung squamous carcinoma cell line, showed synergistic increase in radiosensitivity when cells were pre-treated with HA. In addition, HA significantly inhibited repair of radiation-induced DSBs. These results suggest that the combination of HA and ionizing radiation may be a useful therapeutic strategy for treating certain cancer cells.     

Nucleophilic Cyclopropanation Reaction with Bis(iodozincio)methane by 1,4‐Addition to α,β‐Unsaturated Carbonyl Compounds

Treatment of α,β‐unsaturated ketones with an electrophilic site at the γ‐position in the presence of trimethylsilyl cyanide with bis(iodozincio)methane afforded the (Z)‐silyl enol ether of the β‐cyclopropyl substituted ketone in good yields. The reaction proceeds by 1,4‐addition to form an enolate, and its sequential intramolecular nucleophilic attack to an adjacent electrophilic site. The reaction of γ‐ethoxycarbonyl‐α,β‐unsaturated ketone and bis(iodozincio)methane in the presence of trimethylsilyl cyanide afforded 1‐ethoxy‐1‐trimethylsiloxycyclopropane derivatives, which can be regarded as the homoenolate equivalent. Additionally, reaction of the obtained homoenolate equivalents with imines give 1‐(E)‐alkenyl‐2‐(1‐aminoalkyl)alkanols diastereoselectively.     

Sensitized fluorescence generated in bacterial luciferase catalytic cycle with metal complexbinding fluorescent protein

The Ru(bpy)₃^2+*-MV²⁺ sensitization system formed within a Nafion film was linked with in vitro bacterial luciferase reaction in the presence of a blue fluorescent protein. The electron released from the excited triplet Ru(bpy)₃^2+* was transferred to the luciferase catalytic cycle with the fluorescent protein via electron relay, resulting in sensitization of the fluorescence protein.