Takeshi Kubota

Inhibition of RANKL-induced osteoclastogenesis by (-)-DHMEQ, a novel NF-κB inhibitor, through downregulation of NFATc1

(−)‐DHMEQ, a newly designed NF‐κB inhibitor, inhibited RANKL‐induced osteoclast differentiation in mouse BMMs through downregulation of the induction of NFATc1, an essential transcription factor of osteoclastogenesis.

Sequence Dependence of Fluorescence Emission and Quenching of Doubly Thiazole Orange Labeled DNA: Effective Design of a Hybridization-Sensitive Probe

We have designed a doubly thiazole orange labeled nucleoside showing high fluorescence intensity for a hybrid with the target DNA and effective quenching for a single-stranded state. Knowing how much the fluorescence emission and quenching of this probe depend on the probe sequence and why there is such a sequence dependence is important for effective probe design, we synthesized more than 30 probe sequences and measured their fluorescence intensities. When the probe hybridized with the target DNA strands, there was strong emission, whereas the emission intensity was much weaker before hybridization; however, self-dimerization of probes suppressed fluorescence quenching. In particular, the G/C base pairs neighboring the labeled nucleotide in a self-dimeric structure resulted in a low quenching ability for the probe before hybridization. On the other hand, mismatched base pair formation around the labeled site decreased the fluorescence intensity because the neighboring sequence is the binding site of the tethered thiazole orange dyes. The hybridization enhanced the fluorescence of the probe even when the labeled nucleotide was located at the end of the probe strand; however, the partial lack of duplex structure resulted in a decrease in the fluorescence intensity of the hybrid.

Sets of RNA Repeated Tags and Hybridization-Sensitive Fluorescent Probes for Distinct Images of RNA in a Living Cell

Background Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell. Methodology/Principal Findings Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO) probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3′-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag–probe pairs. Conclusions/Significance A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging.

Na+/Mg2+ transporter acts as a Mg2+ buffering mechanism in PC12 cells

Mg(2+) buffering mechanisms in PC12 cells were demonstrated with particular focus on the role of the Na(+)/Mg(2+) transporter by using a newly developed Mg(2+) indicator, KMG-20, and also a Na(+) indicator, Sodium Green. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a protonophore, induced a transient increase in the intracellular Mg(2+) concentration ([Mg(2+)](i)). The rate of decrease of [Mg(2+)](i) was slower in a Na(+)-free extracellular medium, suggesting the coupling of Na(+) influx and Mg(2+) efflux. Na(+) influxes were different for normal and imipramine- (a putative inhibitor of the Na(+)/Mg(2+) transporter) containing solutions. FCCP induced a rapid increase in [Na(+)](i) in the normal solution, while the increase was gradual in the imipramine-containing solution. The rate of decrease of [Mg(2+)](i) in the imipramine-containing solution was also slower than that in the normal solution. From these results, we show that the main buffering mechanism for excess Mg(2+) depends on the Na(+)/Mg(2+) transporter in PC12 cells.

Design and Synthesis of Caged Fluorescent Nucleotides and Application to Live-cell RNA Imaging

A binary photocontrolled nucleic acid probe that contains a nucleotide modified with one photolabile nitrobenzyl unit and two hybridization‐sensitive thiazole orange units has been designed for area‐specific fluorescence imaging of RNA in a cell. The synthesized probe emitted very weak fluorescence regardless of the presence of the complementary RNA, whereas it showed hybridization‐sensitive fluorescence emission at 532 nm after photoirradiation at 360 or 405 nm for uncaging. Fluorescence suppression of the caged probe was attributed to a decrease in the duplex‐formation ability. Caged fluorescent nucleotides with other emission wavelengths (622 and 724 nm) were also synthesized in this study; they were uncaged by 360 nm irradiation, and emitted fluorescence in the presence of the complementary RNA. Such probes were applied to area‐specific RNA imaging in a cell. Only probes in the defined irradiation area were activated by uncaging irradiation, and subnuclear mRNA diffusion in a living cell was monitored.

Cy5-Conjugated Hybridization-Sensitive Fluorescent Oligonucleotides for Ratiometric Analysis of Nuclear Poly(A)+ RNA

Subnuclear poly(A)(+) RNA localization in living mammalian cells was visualized by ratiometric analysis using hybridization-sensitive fluorescent oligonucleotide probes. Probes were oligonucleotides, which contained a Cy5 fluorescent dye at the strand end and a thiazole orange double-labeled nucleotide inside strand. A ratiometric analysis using poly(A)-targeting probes revealed a distribution of the probe itself as red fluorescence and localization of the target RNA sequence in cell nuclei as green fluorescence. The fluorescence of the subnuclear poly(A)(+) RNA hybridized with the poly(A)-targeting probes was observed as puncta in interchromatin areas.

Synthesis of exciton-controlled fluorescent probes for RNA imaging

The excitonic interaction of thiazole orange dyes is known to suppress fluorescence emission. We have developed hybridization-sensitive fluorescent probes utilizing the excitonic interaction of two thiazole orange dyes connected to the probes. Here, we report nuclease-resistant hybridization-sensitive probes for long-term intracellular RNA imaging.

Investigation of intracellular magnesium mobilization pathways I PC12 cells B simultaneous Mg-Ca fluorescent imaging

Objective and Methods: PC12 cells were loaded with a novel Mg indicator KMG-104 and Ca indicator fura-2, and intracellular Mg was studied in the endoplasmic reticulums (ERs), mitochondria, and Mg-ATP. Under coexistence of the two indicators, fluorescent signals of Mg and Ca can be measured separately. Mg release from the ER was investigated by photolysis of caged compounds. Results: Transient [Ca] i increase by uncaging of caged Ca or caged IP 3 or bath-application of caffeine (10 mM) induced no [Mg] i increase. These results suggest that there is no mechanism for Mg release from the ER through ryanodine receptors or IP 3 receptors. In order to investigate the possibility of Mg release from Mg-ATP by energy consumption, we depleted ATP by oligomycin, an inhibitor of mitochondrial ATP synthase. Treating with oligomycin (4 μM) for several minutes showed no change of [Mg] i and [Ca] i. Conclusions: This result shows that Mg-ATP is not a Mg store. Since, when cells were treated by an uncoupler FCCP (3 μM), [Mg] i and [Ca] i increased, we concluded that mitochondria participate in maintenance of intracellular Mg stores.

Exciton-controlled fluorescence: application to hybridization-sensitive fluorescent DNA probe.

A hybridization-sensitive fluorescent probe has been designed for nucleic acid detection, using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleotide showing high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching for the single-stranded state. This exciton-controlled fluorescent probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence was observed from the probe hybridizing with the target RNA. This fluorescence rapidly decreased upon addition of a competitor DNA. Multicoloring of this probe resulted in the simple simultaneous detection of plural target nucleic acid sequences. This probe realized a large, rapid, reversible change in fluorescence intensity in sensitive response to the amount of target nucleic acid, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.

Intracellular mRNA imaging with a hybridization sensitive fluorescent nucleotide

We reported a technique for detecting intracellular RNA with a newly developed functional probe. To detect mRNAs, a polyA tail targeting probe was synthesized. This probe showed quite large enhancement in the presence of its complementary RNA. In living cultured cells, the probe also showed strong fluorescence. This technique was effective for intracellular mRNA detection.