Synthesis of yellow and red fluorescent 1,3a,6a-triazapentalenes and the theoretical investigation of their optical properties

To expand the originally developed fluorescent 1,3a,6a-triazapentalenes as fluorescent labelling reagents, the fluorescence wavelength of the 1,3a,6a-triazapentalenes was extended to the red color region. Based on the noteworthy correlation of the fluorescence wavelength with the inductive effect of the 2-substituent, electron-deficient 2-(2-cyano-4-methoxycarbonylphenyl)-1,3a,6a-triazapentalene and 2-(2,6-dicyano-4-methoxycarbonylphenyl)-1,3a,6a-triazapentalene were synthesized. The former exhibited yellow fluorescence and the latter exhibited red fluorescence, and both compounds exhibited large Stokes shifts, and the 1,3a,6a-triazapentalene system enabled the same fluorescent chromophore to cover the entire region of visible wavelengths. The potential applications of the 1,3a,6a-triazapentalenes as fluorescent probes in the fields of the life sciences were investigated, and the 1,3a,6a-triazapentalene system was clearly proven to be useful as a fluorescent reagent for live cell imaging. Quantum chemical calculations were performed to investigate the optical properties of the 1,3a,6a-triazapentalenes. These calculations revealed that the excitation involves a significant charge-transfer from the 1,3a,6a-triazapentalene skeleton to the 2-substituent. The calculated absorption and fluorescence wavelengths showed a good correlation with the experimental ones, and thus the system could enable the theoretical design of substituents with the desired optical properties

Cellular Morphology Visualization to Probe Cell Differentiation

Nuclear and cytoplasmic morphological changes provide important information about cell differentiation processes, cell functions, and signal responses. There is a strong desire to develop a rapid and simple method for visualizing cytoplasmic and nuclear morphology. Here, we developed a novel and rapid method for probing cellular morphological changes of live cell differentiation process by a fluorescent probe, TAP-4PH, a 1,3a,6a-triazapentalene derivative. TAP-4PH showed high fluorescence in cytoplasmic area, and visualized cytoplasmic and nuclear morphological changes of live cells during differentiation. We demonstrated that TAP-4PH visualized dendritic axon and spine formation in neuronal differentiation, and nuclear structural changes during neutrophilic differentiation. We also showed that the utility of TAP-4PH for visualization of cytoplasmic and nuclear morphologies of various type of live cells. Our visualizing method has no toxicity and no influence on the cellular differentiation and function. The cell morphology can be rapidly observed after addition of TAP-4PH and can continue to be observed in the presence of TAP-4PH in cell culture medium. Moreover, TAP-4PH can be easily removed after observation by washing for subsequent biological assay. Taken together, these results demonstrate that our visualization method is a powerful tool to probe differentiation processes before subsequent biological assay in live cells

PPM1D controls nucleolar formation by up-regulating phosphorylation of nucleophosmin

An increase of nucleolar number and size has made nucleoli essential markers for cytology and tumour development. However, the underlying basis for their structural integrity and abundance remains unclear. Protein phosphatase PPM1D was found to be up-regulated in different carcinomas including breast cancers. Here, we demonstrate for the first time that PPM1D regulates nucleolar formation via inducing an increased phosphorylation of the nucleolar protein NPM. We show that PPM1D overexpression induces an increase in the nucleolar number regardless of p53 status. We also demonstrated that specific sequential phosphorylation of NPM is important for nucleolar formation and that PPM1D is a novel upstream regulator of this phosphorylation pathway. These results enhance our understanding of the molecular mechanisms that govern nucleoli formation by demonstrating that PPM1D regulates nucleolar formation by regulating NPM phosphorylation status through a novel signalling pathway, PPM1D-CDC25C-CDK1-PLK1

Patterning nanofibrils through the templated growth of multiple modified amyloid peptides

There has been considerable interest in the patterning of functionalized nanowires because of the potential applications of these materials to the construction of nanodevices. A variety of biomolecular building blocks containing amyloid peptides have been used to functionalize nanowires. However, the patterning of self-assembled nanowires can be challenging because of the difficulties associated with controlling the self-assembly of these functionalized building blocks. Herein, we present a versatile approach for the patterning of nanowires based on the combination of templated fibril growth with a versatile functionalization method using our structure-controllable amyloid peptides (SCAPs). Using this approach, we have succeeded in the formation of multi-type nanowires with tandem domain structures in high yields. Given that the mixing-SCAP method can lead to the formation of tandem fibrils, it is noteworthy that our method allowed us to control the initiation of fibril formation from the gold nanoparticles, which were attached to a short fibril as initiation points. This approach could be used to prepare a wide variety of fibril patterns, and therefore holds great potential for the development of novel self-assembled nanodevices

Effects of E/Z Configuration of Fluoroalkene-containing HDAC Inhibitors on Selectivity for HDAC Isoforms

Histone deacetylase (HDAC) inhibitors belong to a new class of potential anticancer agents. It may be possible to reduce some of the toxicity by specifically targeting only the HDAC isoform. Here, stereoisomeric HDAC inhibitors containing fluoroalkene were analyzed for their specificity toward HDAC isoforms. Z-Form 1(Z) showed high affinity to HDACs whereas E-isoform 1(E) had lower affinity to HDAC1 and HDAC4. These data suggested that introduction of alkene with E/Z configuration to HDAC inhibitor can be a new strategy to develop the isoform-selective HDAC inhibitors

Development of Specific Inhibitors for Oncogenic Phosphatase PPM1D by Using Ion-Responsive DNA Aptamer Library

(1) Background: Ser/Thr protein phosphatase PPM1D is an oncogenic protein. In normal cells, however, PPM1D plays essential roles in spermatogenesis and immune response. Hence, it is necessary to develop novel PPM1D inhibitors without side effects on normal cells. Stimuli-responsive molecules are suitable for the spatiotemporal regulation of inhibitory activity. (2) Methods: In this study, we designed an ion-responsive DNA aptamer library based on G-quadruplex DNA that can change its conformation and function in response to monovalent cations. (3) Results: Using this library, we identified the PPM1D specific inhibitor M1D-Q5F aptamer. The M1D-Q5F aptamer showed anti-cancer activity against breast cancer MCF7 cells. Interestingly, the induction of the structural change resulting in the formation of G-quadruplex upon stimulation by monovalent cations led to the enhancement of the inhibitory activity and binding affinity of M1D-Q5F. (4) Conclusions: These data suggest that the M1D-Q5F aptamer may act as a novel stimuli-responsive anti-cancer agent

Effective Cellular Morphology Analysis for Differentiation Processes by a Fluorescent 1,3a,6a-Triazapentalene Derivative Probe in Live Cells.

Nuclear and cytoplasmic morphological changes provide important information about cell differentiation processes, cell functions, and signal responses. There is a strong desire to develop a rapid and simple method for visualizing cytoplasmic and nuclear morphology. Here, we developed a novel and rapid method for probing cellular morphological changes of live cell differentiation process by a fluorescent probe, TAP-4PH, a 1,3a,6a-triazapentalene derivative. TAP-4PH showed high fluorescence in cytoplasmic area, and visualized cytoplasmic and nuclear morphological changes of live cells during differentiation. We demonstrated that TAP-4PH visualized dendritic axon and spine formation in neuronal differentiation, and nuclear structural changes during neutrophilic differentiation. We also showed that the utility of TAP-4PH for visualization of cytoplasmic and nuclear morphologies of various type of live cells. Our visualizing method has no toxicity and no influence on the cellular differentiation and function. The cell morphology can be rapidly observed after addition of TAP-4PH and can continue to be observed in the presence of TAP-4PH in cell culture medium. Moreover, TAP-4PH can be easily removed after observation by washing for subsequent biological assay. Taken together, these results demonstrate that our visualization method is a powerful tool to probe differentiation processes before subsequent biological assay in live cells