論文

Background-Hydrogen sulfide (H2S) exhibits protective effects in various disease models including cerebral ischemia-reperfusion (I/R) injury. Nonetheless, mechanisms and identity of molecules responsible for neuroprotective effects of H2S remain incompletely defined. In the current study, we observed that thiosulfate, an oxidation product of H2S, mediates protective effects of an H2S donor compound sodium sulfide (Na2S) against neuronal I/R injury. Methods and Results-We observed that thiosulfate in cell culture medium is not only required but also sufficient to mediate cytoprotective effects of Na2S against oxygen glucose deprivation and reoxygenation of human neuroblastoma cell line (SH-SY5Y) and murine primary cortical neurons. Systemic administration of sodium thiosulfate (STS) improved survival and neurological function of mice subjected to global cerebral I/R injury. Beneficial effects of STS, as well as Na2S, were associated with marked increase of thiosulfate, but not H2S, in plasma and brain tissues. These results suggest that thiosulfate is a circulating "carrier" molecule of beneficial effects of H2S. Protective effects of thiosulfate were associated with inhibition of caspase-3 activity by persulfidation at Cys163 in caspase-3. We discovered that an SLC13 family protein, sodium sulfate cotransporter 2 (SLC13A4, NaS-2), facilitates transport of thiosulfate, but not sulfide, across the cell membrane, regulating intracellular concentrations and thus mediating cytoprotective effects of Na2S and STS. Conclusions-The protective effects of H2S are mediated by thiosulfate that is transported across cell membrane by NaS-2 and exerts antiapoptotic effects via persulfidation of caspase-3. Given the established safety track record, thiosulfate may be therapeutic against ischemic brain injury.

Superior fluorescence imaging methods are needed for detailed studies on biological phenomena, and one approach that permits precise analyses is time-resolved fluorescence measurement, which offers a high signal-to-noise ratio. Herein, we describe a new fluorescence imaging system to visualize biomolecules within living biological samples by means of time-resolved, long-lived luminescence microscopy (TRLLM). In TRLLM, short-lived background fluorescence and scattered light are gated out, allowing the long-lived luminescence to be selectively imaged. Usual time-resolved fluorescence microscopy provides fluorescence images with nanosecond resolution and has been used to image interactions between proteins, protein phosphorylation, the local pH, the refractive index, ion or oxygen concentrations, etc. Luminescent lanthanide complexes (especially europium and terbium trivalent ions (Eu 3+ and Tb3+)), in contrast, have long luminescence lifetimes on the order of milliseconds. We have designed and synthesized new luminescent Eu3+ complexes for TRLLM and also developed a new TRLLM system using a conventional fluorescence microscope with an image intensifier unit for gated signal acquisition and a xenon flash lamp as the excitation source. When the newly developed luminescent Eu3+ complexes were applied to living cells, clear fluorescence images were acquired with the TRLLM system, and short-lived fluorescence was completely excluded. By using Eu 3+ and Tb3+ luminescent complexes in combination, time-resolved dual-color imaging was also possible. Furthermore, we monitored changes of intracellular ionic zinc (Zn2+) concentration by using a Zn2+-selective luminescent Eu3+ chemosensor, [Eu-7]. This new imaging technique should facilitate investigations of biological functions with fluorescence microscopy, complementing other fluorescence imaging methodologies. © 2007 American Chemical Society.

Investigation of the unexpected photo-instability of 2,6-sulfonamide-substituted derivatives of the boron dipyrromethene (BODIPY) fluorophore led to the discovery of a photoreaction accompanied by multiple bond scissions. We characterized the photoproducts and utilized the photoreaction to design a caged γ-aminobutyric acid (GABA) derivative that can release GABA upon irradiation in the visible range (>450 nm). This allowed us to stimulate neural cells in mouse brain slices.

A linear ubiquitin chain, which consists of ubiquitin molecules linked via their N- and C-termini, is formed by a linear ubiquitin chain assembly complex (LUBAC) composed of HOIP, HOIL-1L, and SHARPIN, and conjugation of a linear ubiquitin chain on the NF-κB essential modulator (NEMO) is deeply involved in NF-κB activation induced by various signals. Since abnormal activation of NF-κB is associated with inflammatory disease and malignancy, we searched for an inhibitor of LUBAC by high-throughput screening (HTS) with a Tb3+-fluorescein FRET system. As a result, we found that the fungal metabolite gliotoxin inhibits LUBAC selectively by binding to the RING-IBR-RING domain of HOIP, the catalytic center of LUBAC. Gliotoxin has been well-known as an inhibitor of NF-κB activation, though its action mechanism has remained elusive. Here, we show that gliotoxin inhibits signal-induced NF-κB activation by selectively inhibiting LUBAC-mediated linear ubiquitin chain formation.

Optical highlighters are photoactivatable fluorescent molecules that exhibit pronounced changes in their spectral properties in response to irradiation with light of a specific wavelength and intensity. Here, we present a novel design strategy for a new class of caged BODIPY (4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene) fluorophores, based on the use of photoremovable protecting groups (PRPGs) with high reduction potentials that serve as both a photosensitive unit and a fluorescence quencher via photoinduced electron transfer (PeT). 2,6-Dinitrobenzyl (DNB)-caged BODIPY was efficiently photoactivated, with activation ratios exceeding 600-fold in aqueous solutions. We then combined this photoactivatable fluorophore with a SNAP (mutant of O 6-alkylguanine DNA alkyltransferase) ligand to obtain a small-molecule-based optical highlighter for visualization of protein dynamics, using the well-established SNAP tag technology. As proof of concept, we demonstrate spatiotemporal imaging of the fusion protein of epidermal growth factor receptor (EGFR) with SNAP tag in living cells. We also demonstrate highlighting of cells of interest in live zebrafish embryos, using the fusion protein of histone 2A with SNAP tag. © 2012 American Chemical Society.

PEG-coated β-FeOOH nanoparticles were prepared through electrostatic complex formation of iron oxide nanoparticles with poly(ethylene glycol)-poly(aspartic acid) block copolymer [PEG-P(Asp)] in distilled water. By dynamic light scattering (DLS) measurement, the nanopaticle size was determined to be 70 nm with narrow distribution. The FT-IR and zeta potential experimental results proved that PEG-PAsp molecules bound to the surface of the iron oxide nanoparticles via the coordination between the carboxylic acid residues in the PAsp segment of the block copolymer and the surface Fe of the β-FeOOH nanoparticles. The PEG-coated nanoparticles revealed excellent solubility and stability in aqueous solution as well as in physiological saline. In vivo MRI experiments on tumor-bearing mice demonstrated that the PEG-coated nanoparticles prepared by the current approach achieved an appreciable accumulation into solid tumor, suggesting their potential utility as tumor-selective MRI contrast agents. © 2007 Elsevier B.V. All rights reserved.

Gadolinium ion (Gd 3+) complexes are commonly used as magnetic resonance imaging (MRI) contrast agents to enhance signals in T 1-weighted MR images. Recently, several methods to achieve cell-permeation of Gd 3+ complexes have been reported, but more general and efficient methodology is needed. In this report, we describe a novel method to achieve cell permeation of Gd 3+ complexes by using hydrophobic fluorescent dyes as a cell-permeability-enhancing unit. We synthesized Gd 3+ complexes conjugated with boron dipyrromethene (BDP-Gd) and Cy7 dye (Cy7-Gd), and showed that these conjugates can be introduced efficiently into cells. To examine the relationship between cell permeability and dye structure, we further synthesized a series of Cy7-Gd derivatives. On the basis of MR imaging, flow cytometry, and ICP-MS analysis of cells loaded with Cy7-Gd derivatives, highly hydrophobic and nonanionic dyes were effective for enhancing cell permeation of Gd 3+ complexes. Furthermore, the behavior of these Cy7-Gd derivatives was examined in mice. Thus, conjugation of hydrophobic fluorescent dyes appears to be an effective approach to improve the cell permeability of Gd 3+ complexes, and should be applicable for further development of Gd 3+-based MRI contrast agents. © 2011 American Chemical Society.

In spite of the wide availability of various near-infrared (NIR) fluorophores as labeling reagents, there are few functional NIR fluorescent probes for which change in the absorption and/or fluorescence spectra upon specific reaction with biomolecules is seen. The widely used photoinduced electron-transfer mechanism is unsuitable for NIR fluorophores, such as tricarbocyanines, because their long excitation wavelength results in a small singlet ex-citation energy. We have reported the unique spectral properties of aminesubstituted tricarbocyanines, which were utilized to develop two design strategies. One approach was based on control of the absorption wavelength by using the difference in electron-do-nating ability before and after a specific reaction with a biomolecule, and the other approach was based on control of the fluorescence intensity by modulating the Förster resonance energytransfer efficiency through a change in the overlap integral that arises from the change in absorption under acidic conditions. These strategies were validated by obtaining tricarbocyaninebased ratiometric NIR fluorescent probes for esterase and for pH level. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA,.

The cysteine prodrug N-acetyl cysteine (NAC) is widely used as a pharmacological antioxidant and cytoprotectant. It has been reported to lower endogenous oxidant levels and to protect cells against a wide range of pro-oxidative insults. As NAC itself is a poor scavenger of oxidants, the molecular mechanisms behind the antioxidative effects of NAC have remained uncertain. Here we show that NAC-derived cysteine is desulfurated to generate hydrogen sulfide, which in turn is oxidized to sulfane sulfur species, predominantly within mitochondria. We provide evidence suggesting the possibility that sulfane sulfur species produced by 3-mercaptopyruvate sulfurtransferase and sulfide:quinone oxidoreductase are the actual mediators of the immediate antioxidative and cytoprotective effects provided by NAC. N-Acetyl cysteine (NAC), by itself a poor scavenger of oxidants, is converted inside cells to yield sulfane sulfur species, which are very potent scavengers of oxidants. This conversion may account for many of the antioxidative effects provided by NAC which have hitherto remained unexplained.

Hydrogen sulfide (H2S) exerts synergistic effects with another gaseous signaling molecule nitric oxide (NO) on ion channels and vasculature. However, the mechanism of the synergy is not well understood. Here, we show that the interaction between H2S and NO generates polysulfides (H2Sn), which activate transient receptor potential ankyrin 1 (TRPA1) channels. High performance liquid chromatography with tandem mass spectrometry analysis, along with the imaging of intracellular Ca2+ and H2Sn, showed that H2Sn and their effects were abolished by cyanolysis and by reducing substances such as dithiothreitol (DTT), cysteine, and glutathione (GSH). However, the effects of nitroxyl or nitrosopersulfide, other potential products of H2S and NO interaction, are not affected by cyanolysis or reducing substances. This study demonstrates that H2Sn are products of synergy between H2S and NO and provides a new insight into the signaling mechanisms.

The oxidation of cysteine in mammalian cells occurs by two routes: a highly regulated direct oxidation pathway in which the first step is catalyzed by cysteine dioxygenase (CDO) and by desulfhydration-oxidation pathways in which the sulfur is released in a reduced oxidation state. To assess the effect of a lack of CDO on production of hydrogen sulfide (H2S) and thiosulfate (an intermediate in the oxidation of H2S to sulfate) and to explore the roles of both cystathionine γ-lyase (CTH) and cystathionine β-synthase (CBS) in cysteine desulfhydration by liver, we investigated the metabolism of cysteine in hepatocytes isolated from Cdo1-null and wild-type mice. Hepatocytes from Cdo1-null mice produced more H2S and thiosulfate than did hepatocytes from wild-type mice. The greater flux of cysteine through the cysteine desulfhydration reactions catalyzed by CTH and CBS in hepatocytes from Cdo1-null mice appeared to be the consequence of their higher cysteine levels, which were due to the lack of CDO and hence lack of catabolism of cysteine by the cysteinesulfinate-dependent pathways. Both CBS and CTH appeared to contribute substantially to cysteine desulfhydration, with estimates of 56 % by CBS and 44 % by CTH in hepatocytes from wild-type mice, and 63 % by CBS and 37 % by CTH in hepatocytes from Cdo1-null mice. © 2014 Springer-Verlag Wien.

K+ is the most abundant metal ion in cells, and changes of [K+] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K+] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K+, TLSHalo. TLSHalo is responsive to [K+] in the physiological range, with good selectivity over Na+ and retains its K+-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K+] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K+] change induced by K+ ionophores or by activation of a native Ca2+-dependent K+ channel (BK channel). Further, we show that K+ efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K+ dynamics with high spatiotemporal resolution.

Fluorescence imaging in the near-infrared (NIR) region (650–900 nm) is useful for bioimaging because background autofluorescence is low and tissue penetration is high in this range. In addition, NIR fluorescence is useful as a complementary color window to green and red for multicolor imaging. Here, we compared the photoinduced electron transfer (PeT)-mediated fluorescence quenching of silicon- and phosphorus-substituted rhodamines (SiRs and PRs) in order to guide the development of improved far-red to NIR fluorescent dyes. The results of density functional theory calculations and photophysical evaluation of a series of newly synthesized PRs confirmed that the fluorescence of PRs was more susceptible than that of SiRs to quenching via PeT. Based on this, we designed and synthesized a NIR fluorescence probe for Ca2+, CaPR-1, and its membrane-permeable acetoxymethyl derivative, CaPR-1 AM, which is distributed to the cytosol, in marked contrast to our previously reported Ca2+ far-red to NIR fluorescence probe based on the SiR scaffold, CaSiR-1 AM, which is mainly localized in lysosomes as well as cytosol in living cells. CaPR-1 showed longer-wavelength absorption and emission (up to 712 nm) than CaSiR-1. The new probe was able to image Ca2+ at dendrites and spines in brain slices, and should be a useful tool in neuroscience research.

We studied the photobleaching of a library of boron dipyrromethene (BODIPY) derivatives with a range of electron densities, and found that the photobleaching rate is influenced by the electron-withdrawing capacity of the substituents. Electron-deficient BODIPYs generated less singlet oxygen, were less reactive to singlet oxygen, and were highly resistant to photobleaching. We confirmed the utility of one of these fluorophores, 2,6-diCO2R-BDP, for visualizing EGF receptor dynamics in cells expressing an SNAP-tagged EGF receptor. © The Royal Society of Chemistry 2011.

Novel ratiometric, near-infrared fluorescent pH probes with various pK a values have been designed and synthesized on the basis of aminocyanine bearing a diamine moiety, and their photochemical properties were evaluated. Under acidic conditions, these pH probes showed a 46- to 83-nm red shift of the absorption maximum. This change is sufficiently large to permit their use as ratiometric pH probes, and is reversible, whereas monoamine-substituted aminocyanines showed irreversible changes because of their instability under acidic conditions. Furthermore, the pKa values of these probes can be predicted from the calculated pKa values of the diamine moieties, obtained from the SciFinder database. This design strategy is very simple and flexible, and should be applicable to develop NIR pH probes for various applications. © 2011 American Chemical Society.

Adenosine 5’ triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF1-ATP synthase e subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.

We see red (and yellow and green): Probe 1 was developed for the visualization of cytoplasmic Ca2+, a pivotal second messenger in many biological responses. The new probe is suitable for multicolor imaging for the simultaneous detection of metal ions or proteins and is superior to the existing red fluorescent probe Rhod-2 for the monitoring of cytoplasmic Ca2+ oscillation in cultured cells (see fluorescence images of cells with 1 (top) and Rhod-2 (bottom)). © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.