Luminol-hydrogen peroxide-horseradish peroxidase chemiluminescence intensification by kosmotrope ammonium sulfate.

The kosmotropic effect induced by ammonium sulfate (AS) at concentrations greater than approximately 2.8 M allows the marked intensification of chemiluminescence (CL) arising from a conventional luminol-hydrogen peroxide (H2O2)-horseradish peroxidase (HRP) reaction. Because of the kosmotropic effect, CL is intensified by at least three orders of magnitude than that from the conventional HRP-catalyzed luminol reaction with no AS; the linear relationship between the CL intensity and the HRP concentration is established over the range of 0.3 pM to several tens of pM. The novel CL intensification effect on the HRP-catalyzed luminol CL can be stably and reproducibly induced.

Direct electron transfer of Cellulomonas fimi and microbial fuel cells fueled by cellulose.

The strain of Cellulomonas fimi NBRC 15513 can generate electricity with cellulose as fuel without mediator using a single chamber type microbial fuel cell (MFC) which had 100 mL of chamber and 50 cm2 of the air cathode. The MFCs were operated over five days and showed the maximum current density of 10.0 ± 1.8 mA/m2, the maximum power density of 0.74 ± 0.07 mW/m2 and the ohmic resistance of 6.9 kΩ. According to the results of cyclic voltammetry, the appearance of the oxidation or reduction peak was not observed from the cell removed solution. The fact is that C. fimi does not secrete mediator-like compounds, while the oxidation peak was observed at +0.68 V from the phosphate buffer containing the washed cell. The peak appearance was caused by the electron transfer activity of which corresponds to cytochrome c, and disappeared after adding antimycin A which inhibits the electron transfer activity. The cell was alive throughout the experiment as the result of a colony forming unit on Luria-Bertani agar plates. This was thought that cytochrome c was on the membrane surface of the living cell and played a role in the direct electron transfer between the cells and anode.

Bioluminescence Microplate Assay of Cyanide with Escherichia coli Harboring a Plasmid Responsible for Cyanide-dependent Light Emission in Alginate Microenvironment.

We describe the bioluminescence of a genetically engineered Escherichia coli harboring a recombined plasmid with a catalase gene promoter fused lux gene cluster, responsible for the generation of photons closely associated with respiratory inhibition, with the aim of applying it for cyanide sensing. This E. coli construct was favorably utilized for the microplate assay of cyanide by leveraging the microenvironment of the biocompatible alginate. The brightness of the bioluminescence, induced by cyanide stimulation of the respiration causative of the production of hydrogen peroxide, positively correlates with its concentration. Moreover, visualization of cyanide with a consumer digital camera, ranging in concentration from about 0.01 mg CN·L-1 in the alginate sol to around 100 mg CN·L-1 in its gel, was attained.

Visualization of mitochondria in living cells with a genetically encoded yellow fluorescent protein originating from a yellow-emitting luminous bacterium.

We have visualized redox and structural changes in the mitochondria of yeast Saccharomyces cerevisiae as a eukaryotic cell model using a genetically encoded yellow fluorescent protein (Y1-Yellow) and conventional fluorescence microscopy. Y1-Yellow originating from a yellow emitting luminous bacterium Aliivibrio sifiae Y1 was fused with a mitochondria-targeted sequence (mt-sequence). Y1-Yellow fluorescence arising only from the mitochondrial site and the color of yellow fluorescence could be easily differentiated from cellular autofluorescence and from that of conventional probes. Y1-Yellow expressing S. cerevisiae made the yellow fluorescence conspicuous at the mitochondrial site in response to reactive oxygen species (ROS) transiently derived in the wake of pretreatment with hydrogen peroxide. Based on our observation with Y1-Yellow fluorescence, we also showed that mitochondria rearrange to form a cluster structure surrounding chromosomal DNA via respiratory inhibition by cyanide, followed by the generation of ROS. In contrast, uptake of an uncoupler of oxidative phosphorylation is not responsible for mitochondrial rearrangement. These results indicate the utility of Y1-Yellow for visualization of mitochondrial vitality and morphology in living cells.

Characterization of protein-like fluorophores released from lake phytoplankton on the basis of fractionation and electrophoresis.

Three kinds of lake plankton were cultivated, and the properties of protein-like fluorophores released from the plankton were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results were compared with those by gel chromatography with a fluorescence detector and three-dimensional excitation-emission matrix (3-DEEM). The concentrated protein-like fluorophores of algal dissolved organic matter (DOM) were successfully separated from the fulvic-like fluorophores, and analyzed using SDS-PAGE. SDS-PAGE analysis revealed that the protein-like fluorescence DOM released from Microcystis aeruginosa consisted of proteins with molecular weights of 17, 37, 50, 75, 150 kDa, and greater than 250 kDa. The results of SDS-PAGE were consistent with those of gel chromatography. Those substances with molecular weights greater than 250 kDa may be a polysaccharide-peptide complex, called peptidoglycan, which is a component of bacterial cell walls. The molecular weights of protein-like fluorescence DOM from Staurastrum dorsidentiferum were determined to be 37 and 50 kDa. For Cryptomonas ovata, its DOM was found to be composed of substances with molecular weights of between 10 and 150 kDa. The results by high-performance size exclusion chromatography with chemiluminescent nitrogen detection (HPSEC/CLND) analysis suggest that the protein-like fluorophores from the plankton might be composed of substances containing organic nitrogen.

Vibrio azureus emits blue-shifted light via an accessory blue fluorescent protein.

Luminous marine bacteria usually emit bluish-green light with a peak emission wavelength (λ(max) ) at about 490 nm. Some species belonging to the genus Photobacterium are exceptions, producing an accessory blue fluorescent protein (lumazine protein: LumP) that causes a blue shift, from λ(max)  ≈ 490 to λ(max)  ≈ 476 nm. However, the incidence of blue-shifted light emission or the presence of accessory fluorescent proteins in bacteria of the genus Vibrio has never been reported. From our spectral analysis of light emitted by 16 luminous strains of the genus Vibrio, it was revealed that most strains of Vibrio azureus emit a blue-shifted light with a peak at approximately 472 nm, whereas other Vibrio strains emit light with a peak at around 482 nm. Therefore, we investigated the mechanism underlying this blue shift in V. azureus NBRC 104587(T) . Here, we describe the blue-shifted light emission spectra and the isolation of a blue fluorescent protein. Intracellular protein analyses showed that this strain had a blue fluorescent protein (that we termed VA-BFP), the fluorescent spectrum of which was almost identical to that of the in vivo light emission spectrum of the strain. This result strongly suggested that VA-BFP was responsible for the blue-shifted light emission of V. azureus.

Crystallization and preliminary crystallographic studies of the HA3 subcomponent of the type B botulinum neurotoxin complex.

The haemagglutinin subcomponent HA3 of the type B botulinum neurotoxin complex, which is important in toxin absorption from the gastrointestinal tract, has been expressed, purified and subsequently crystallized in two crystal forms at different pH values. Form I belonged to space group R32, with unit-cell parameters a = b = 357.4, c = 249.5 Å, α = ß = 90, γ = 120°. Form II belonged to space group I4(1)32, with unit-cell parameters a = b = c = 259.0 Å, α = ß = γ = 90°. Diffraction data were collected from these crystals to a resolution of 3.0 Å for both form I and form II.

Crystal structure of Clostridium perfringens enterotoxin displays features of beta-pore-forming toxins.

Clostridium perfringens enterotoxin (CPE) is a cause of food poisoning and is considered a pore-forming toxin, which damages target cells by disrupting the selective permeability of the plasma membrane. However, the pore-forming mechanism and the structural characteristics of the pores are not well documented. Here, we present the structure of CPE determined by x-ray crystallography at 2.0 Å. The overall structure of CPE displays an elongated shape, composed of three distinct domains, I, II, and III. Domain I corresponds to the region that was formerly referred to as C-CPE, which is responsible for binding to the specific receptor claudin. Domains II and III comprise a characteristic module, which resembles those of ß-pore-forming toxins such as aerolysin, C. perfringens ε-toxin, and Laetiporus sulfureus hemolytic pore-forming lectin. The module is mainly made up of ß-strands, two of which span its entire length. Domain II and domain III have three short ß-strands each, by which they are distinguished. In addition, domain II has an α-helix lying on the ß-strands. The sequence of amino acids composing the α-helix and preceding ß-strand demonstrates an alternating pattern of hydrophobic residues that is characteristic of transmembrane domains forming ß-barrel-made pores. These structural features imply that CPE is a ß-pore-forming toxin. We also hypothesize that the transmembrane domain is inserted into the membrane upon the buckling of the two long ß-strands spanning the module, a mechanism analogous to that of the cholesterol-dependent cytolysins.

First observation of luminescence from tris(2,2'-bipyridine)ruthenium(II) triggered by anodic oxidation of oligodeoxyribonucleotide.

Anodic oxidation of oligodeoxyribonucleotide in an alkaline aqueous medium containing tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)3(2+)) was shown to cause luminescence around +1.3 V (vs. Ag/AgCl) with a maximal intensity at approximately 600 nm, possibly originating from Ru(bpy)3(2+) in the d-pi* triplet state. A pivotal initial stage in the light production path was postulated to be the anodic oxidation of 2-deoxyribose residue. This reaction seems to be available for the determination of sub-micromol dm(-3) levels of oligodeoxyribonucleotide.

Relationship between the redox change in yellow fluorescent protein of Vibrio fischeri strain Y1 and the reversible change in color of bioluminescence in vitro.

To elucidate the reversible change in the color of bioluminescence (BL) arising from Vibrio fischeri Y1, the relationship between the BL color and the redox state of endogenous yellow fluorescent protein (YFP), carrying riboflavin 5'-phosphate (FMN), has been investigated in vitro. YFP lost fluorescence with a maximum at 538 nm when reduced, and retrieved its original fluorescence upon reoxidation. Such a change in YFP fluorescence was analogous to that of free FMN. In the NADH/FMN oxidoreductase-coupled luciferase reaction with YFP, yellow BL peaking around 535 nm was largely depressed when sodium dithionite was added. This phenomenon can be attributed to the reduction of YFP; i.e., reduced YFP does not participate in the luciferase reaction as a secondary emitter. On admitting air into the reaction mixture, the yellow light characteristic of V. fischeri Y1 BL was regenerated. These results indicate that the reversible change in YFP fluorescence is caused by the redox change of YFP-bound FMN, and that the change in BL color between blue and yellow is associated with the redox state of YFP.

LuxA gene of light organ symbionts of the bioluminescent fish Acropoma japonicum (Acropomatidae) and Siphamia versicolor (Apogonidae) forms a lineage closely related to that of Photobacterium leiognathi ssp. mandapamensis.

A molecular phylogenetic analysis of luxA gene sequences of light organ symbionts of the fish Acropoma japonicum (Acropomatidae) and Siphamia versicolor (Apogonidae) revealed that the sequences were related to those of Photobacterium leiognathi ssp. mandapamensis, which is not known to occur as a light organ symbiont among bioluminescent P. leiognathi clades. The presence of another lux gene element, luxF, coding for nonfluorescent protein, provided additional support for the identity of the light organ symbionts of the fish. Cladogenesis of the light organ symbiont P. leiognathi may be influenced by the radiation of host fishes.

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.

Oxygen triggering reversible modulation of Vibrio fischeri strain Y1 bioluminescence in vivo.

Yellow-emitting Vibrio fischeri Y1 modulates its bioluminescence (BL) depending on the dissolved O2 concentration. On supplying O2 to the cells under anaerobiosis, the cells begin to emit striking yellow BL peaking around 535 nm. The enhanced yellow emission reverts reversibly to the original level after O2 is consumed. Moreover, the reversible rise and fall of the yellow emission occurs repeatedly in accord with the repeating cycles of aeration on and off. This indicates that an increase in the cellular amount of yellow fluorescent protein (YFP) is not an immediate cause of the yellow emission enhancement. One suggested explanation is that the activity of YFP originating from its highly fluorescent property is altered by redox interaction with the respiratory components, including the soluble cytochrome c. Under the O2-limited conditions, the cellular YFP molecules, in part, seem to lose the fluorescent property possibly because of being reduced via redox interaction with some respiratory components in reduced form. On stimulating aerobic respiration with O2 supply, the reduced YFP seems to retrieve its fluorescent property via oxidation possibly with both O2, diffused across the cell membrane, and ferricytochrome c, generated during the respiratory turnover. The suggested redox interactions seem primarily to cause the reversible BL modulation.

Voltammetric and spectroelectrochemical characterization of a water-soluble viologen polymer and its application to electron-transfer mediator for enzyme-free regeneration of NADH.

A water-soluble polyxylylviologen (PXV(2+)) was characterized with a view to making use of it as a redox electron-transfer (ET) mediator. Cyclic voltammetric and spectropotentiometric studies showed (i) that PXV(2+) gives two redox waves centering at -0.40 and -0.83 V (vs. Ag/AgCl (3.3 mol dm(-3) KCl)) and (ii) that the lifetime of its monocation radical (PXV(+.)) is two orders of magnitude greater than that of the well-utilized dimethyl viologen monocation radical. Subsequently, the reaction of the PXV(2+/+.) couple with NAD(+) was evaluated in the similar manners as above. On the basis of this evaluation and the bioluminescence assay using bacterial NADH/FMN oxidoreductase and luciferase, it was shown (i) that the PXV(2+/+.) couple functions as a useful electron-transfer mediator and (ii) that PXV(+.) reacts with NAD(+), leading to generation of the enzymatically active NADH, in the absence of any reductases.