Cyclic ferrocenylnaphthalene diimides as a probe for electrochemical telomerase assay.

Novel cyclic naphthalene diimides, 8 and 12, containing ferrocene in the cyclic linker were synthesized as G-quartet (G4) specific electrochemical ligands via the reaction of 1,1'-ferrocenedipropanoic acid and the terminal amine moieties of naphthalene diimides with varying linker lengths. The redox potentials of 8 and 12 were ca. 0.2 V (vs. Ag/AgCl), and the background current in an electrolyte was successfully suppressed. Both 8 and 12 bound to TA-core, representing human telomere G4, with K = 4.4 and 38 × 105 M-1, respectively. The current response of 12 to an electrode immobilized with G4 was the highest among the acyclic derivatives, suggesting its potential application in electrochemical telomerase assays.

Contact Dermatitis Caused by Efinaconazole and Luliconazole.

We report a case of contact dermatitis caused by both efinaconazole, a topical triazole antifungal drug, and luliconazole, a topical imidazole antifungal drug. Positive patch test reactions were observed with efinaconazole and luliconazole. A patch test with lanoconazole also elicited a positive reaction. We hypothesized that structural similarity between luliconazole and lanoconazole led to cross-reaction, and that the dithiolane ring common to both drugs or the structure of the vinyl imidazole with a dithiolane ring could be the antigenic determinant. Since efinaconazole and luliconazole have no common structures, patients could be sensitized to both drugs separately. The antigenic determinant of efinaconazole is unknown. However, the chemical formula of ravuconazole, an oral triazole antifungal drug, is similar to that of efinaconazole. Clinicians should carefully consider potential cross-reactivity between these drugs.

Cyclic Naphthalene Diimide with a Ferrocene Moiety as a Redox-Active Tetraplex-DNA Ligand.

Cyclic naphthalene diimides (cNDIs), with a ferrocene moiety (cFNDs) and different linker lengths between the ferrocene and cNDI moieties, were designed and synthesized as redox-active, tetraplex-DNA ligands. Intramolecular stacking was observed between ferrocene and the NDI planes, which could affect the binding properties for G-quadruplexes. Interestingly, the circular dichroism spectrum of one of these compounds clearly shows new Cotton effects around 320-380 and 240 nm, which can be considered a direct evidence of intramolecular stacking of ferrocene and the NDI. Regarding recognition of hybrid G-quadruplexes, the less rigid structures (longer linkers) show higher binding affinity (106 m-1 order of magnitude). All new compounds show higher selectivity for G4 during electrochemical detection than noncyclic FND derivatives, which further identifies the redox-active potentiality of the cFNDs. Two of the three compounds tested even show preferential inhibition of cell growth in cancer cells over normal cells in a low concentration range, highlighting the potential for bioapplications of these cFNDs.

A Case of Infundibulocystic Basal Cell Carcinoma Clinically Mimicking a Melanocytic Nevus Associated with Epidermal Cysts.

A 52-year-old Japanese woman presented with a 1.5-cm black, glossy, flat, pediculated lump that clinically mimicked a melanocytic nevus on the left temporal side of her head. The subcutaneous tumor beneath the nodule was elastic and hard. A histological examination showed that the tumor was well circumscribed with an exo- and endophytic growth 2.4 × 1.9 cm in size. The lesion contained several keratinous cysts and was composed of funicular fascicles containing squamoid cells. Excessive mucinous material deposition was observed around the tumor periphery and a palisading arrangement of nuclei in the tumor periphery was seen in some areas. Based on these findings, a diagnosis of infundibulocystic basal cell carcinoma (IFC-BCC) was made. This report presents a case of IFC-BCC that clinically mimicked a melanocytic nevus and was also associated with epidermal cysts.

Bcl-XL-binding helical peptides possessing d-Ala residues at their C-termini with the advantage of long-lasting intracellular stabilities.

We attached d-Ala residues to cross-linked helical peptides based on the pro-apoptotic protein Bad at their C-termini. The d-Ala attachment had little influence on the secondary structures and binding abilities against Bcl-XL. The d-Ala attached helical peptides were much more stable in cells than original ones and efficiently induced apoptosis of the cells.

Cyclodextrin-Isolated Alkynylpyrenes as UV-Stable and Blue-Light-Emitting Molecules Even in Condensed States.

Encapsulation of highly emissive alkynylpyrenes with permethylated α-cyclodextrin (PM-α-CD) followed by capping reaction yielded alkynylpyrene-based [3]rotaxanes. The [3]rotaxane emitted only blue light of monomeric pyrene under various circumstances such as lipophilic, hydrophilic, and even condensed states and exhibited extremely high stability for UV irradiation. These properties would result because PM-α-CD, like bulletproof glass, protected the alkynylpyrene core from the attack of another excited alkynylpyrene and singlet oxygen generated by the energy transfer from the excited alkynylpyrene.

Contribution of pannexin 1 and connexin 43 hemichannels to extracellular calcium-dependent transport dynamics in human blood-brain barrier endothelial cells.

Dysregulation of blood-brain barrier (BBB) transport function is thought to exacerbate neuronal damage in acute ischemic stroke. The purpose of this study was to clarify the characteristics of pannexin (Px) and/or connexin (Cx) hemichannel(s)-mediated transport of organic anions and cations in human BBB endothelial cell line hCMEC/D3 and to identify inhibitors of hemichannel opening in hCMEC/D3 cells in the absence of extracellular Ca(2+), a condition mimicking acute ischemic stroke. In the absence of extracellular Ca(2+), the cells showed increased uptake and efflux transport of organic ionic fluorescent dyes. Classic hemichannel inhibitors markedly inhibited the enhanced uptake and efflux. Quantitative targeted absolute proteomics confirmed Px1 and Cx43 protein expression in plasma membrane of hCMEC/D3 cells. Knockdown of Px1 and Cx43 with the small interfering RNAs significantly inhibited the enhanced uptake and efflux of organic anionic and cationic fluorescent dyes. Clinically used cilnidipine and progesterone, which have neuroprotective effects in animal ischemia models, were identified as inhibitors of hemichannel opening. These findings suggest that altered transport dynamics at the human BBB in the absence of extracellular Ca(2+) is at least partly attributable to opening of Px1 and Cx43 hemichannels. Therefore, we speculate that Px1 and Cx43 may be potential drug targets to ameliorate BBB transport dysregulation during acute ischemia.

A doubly alkynylpyrene-threaded [4]rotaxane that exhibits strong circularly polarized luminescence from the spatially restricted excimer.

The Sonogashira coupling of γ-CD-encapsulated alkynylpyrenes with terphenyl-type stopper molecules gave a doubly alkynylpyrene-threaded [4]rotaxane. The rotaxane showed only excimer emission, with a high fluorescence quantum yield of Φf =0.37, arising from the spatially restricted excimer within the cavity of the γ-CD. The excimer emission suffered little from self-quenching up to a concentration of 1.5×10(-5) M and was circularly polarized with a high glum  value of -1.5×10(-2) . The strong circularly polarized luminescence may result from the two stacked pyrenes existing in the rotaxane in an asymmetrically twisted manner.

Tailor-made designer helical peptides that induce mitochondrion-mediated cell death without necrosis.

Managing protein-protein interactions is essential for resolving unknown biological events at the molecular level and developing drugs. We have designed and synthesized a side-chain-crosslinked helical peptides based on the binding domain of a pro-apoptotic protein (Bad) that induces programmed cell death. The peptide showed high helical content and bound to its target, Bcl-XL, more strongly than its non-crosslinked counterparts. When HeLa cells were incubated with the crosslinked peptide, the peptide entered the cytosol across the plasma membrane. The peptide formed a stable complex with Bcl-XL localized at the outer mitochondrial membrane, and this binding event caused the release of cytochrome c from the intermembrane space of mitochondria into the cytosol. This activated the caspase cascade: 70% of HeLa cells died by the apoptosis pathway (without evidence of necrosis).

Treatment for crusted scabies: limitations and side effects of treatment with ivermectin.

Skin eruption with mild itching of the hands and feet developed in a man in his 90s 1 month after he was hospitalized following a traffic accident. Scabies was diagnosed in an attending nurse 3 months after the patient's hospitalization, and infection from the patient was suspected. Cornification of the patient's soles and marked hypertrophy of the nails of both feet were observed. After a large number of scabies mites were detected on microscopic examination, crusted scabies was diagnosed. The patient was given oral ivermectin, 6 mg, once per week for 3 weeks, and crotamiton topical ointment containing 30% benzyl benzoate was applied on the body from the neck down. However, because a large number of scabies mites were detected again on microscopic examination, the dose of ivermectin was increased to 12 mg and administered 3 times. One week after the sixth dose of ivermectin was administered, hemorrhagic scabs around the mouth and erosion of the tongue developed. Mucosal drug eruption was suspected, and eruptions around the mouth and on the tongue resolved within 1 week after ivermectin being discontinued. 1% gamma-benzene hexachloride ointment was applied topically on the body from the neck down once a week, crotamiton ointment containing benzyl benzoate was applied daily, and the hypertrophic parts of the nails were removed. The patient subsequently achieved a full recovery.

[Two cases of allergies due to Anisakis simplex, positive to specific IgE for Ani s 12 allergen].

We report 2 cases of immediate allergies to Anisakis after ingestion of seafood. In case 1, after ingestion of flatfish, sea bream and mackerel, wheals and dyspnea occurred. Result of ImmunoCAP was class 5 for Anisakis. ELISA for specific IgE showed that the patient serum strongly reacted to Ani s 12. In case 2, after ingestion of flatfish and yellowtail, pruritus and dyspnea occurred. Result of ImmunoCAP was class 6 for Anisakis. ELISA for specific IgE showed that the patient serum reacted to Ani s 1, 4, 6 and 12. In both cases, skin prick tests were negative for suspected seafoods. These data suggests the possibility Ani s 12 is a major allergen of Anisakis allergy besides Ani s 1, 2 and 7. Ani s 12 is an allergen that was first reported in 2011. The reactivity of Ani s 12 specific IgE with ELISA may become useful for the diagnosis of Anisakis allergy.

A supramolecular DNA self-assembly based on ß-cyclodextrin-adamantane complexation as a bioorthogonal sticky end motif.

We propose linear end-to-end assemblies of short DNA duplexes based on ß-cyclodextrin-adamantane complexation. The assembled duplexes exhibited increased Tm values compared with those of the corresponding natural hybrids. Competition experiments with external guest molecules showed a substantial decrease in Tm of the terminal modified duplexes, suggesting the viability of inter-duplex complexation.

Photoswitchable, DNA-binding helical peptides assembled with two independently designed sequences for photoregulation and DNA recognition.

Diarylethene-bridged peptides were developed to photoregulate biomolecular interactions. The peptides are made up of diarylethene-bridged and DNA-binding regions at their N- and C termini, respectively. The two regions could be independently designed and combined as desired. The α-helicities of the peptides were photoregulated in on/off or off/on manners, and the manner depended on the positions of two ornithine (Orn) residues for cross-linking reaction at the diarylethene-bridged region. In the case of the on/off manner, when the diarylethene structure adopted the open form on the peptides, the peptides folded into stable α-helices. Upon UV irradiation, the diarylethene moiety isomerized to its closed form to destabilize the helical structures. Quartz crystal microbalance (QCM) analysis showed that the open isomer strongly associated with a target DNA, as compared with the closed one. When the closed-form peptide existing in the DNA complex was irradiated with a fluorescent lamp in the middle of the QCM monitoring, the frequency change (ΔF) was enhanced by the diarylethene photoisomerization.

Side-chain cross-linked short α-helices that behave like original proteins in biomacromolecular interactions.

We explored the effect of α-helical stabilization upon the binding of short peptides to DNAs. The short peptides were designed according to the binding domains of DNA-binding proteins and were cross-linked between their side chains with diacetylenic or isophthalic cross-linking agents to keep stable α-helices. The binding abilities of the peptides to DNAs were evaluated by fluorescence resonance energy transfer analysis. When a cross-linked peptide based on the homeodomain of the transcription factor was titrated with a target DNA duplex, its dissociation constant (Kd) was calculated to be ~0.5 nM. This value was the double-digit smaller than that of the corresponding non-cross-linked peptide. The cross-linked peptide showed high substrate specificity for DNAs at the same level as the original DNA-binding protein.

[Highly emissive pyrene-based fluorophores and highly sensitive fluorescent sensors using pyrene emission switching].

In this review, I report highly emissive pyrene-based fluorophores and highly sensitive fluorescent sensors using pyrene emission switching. Alkynylpyrene derivatives show fruitful photophysical properties, as compared with pyrene itself: longer absorption and emission wavelengths and high fluorescence quantum yields in the presence of O(2). Alkynylpyrene-based fluorescent probes allow the highly sensitive detection of biomolecules such as proteins. Additionally, a rotaxane-based fluorophore, consisting of an alkynylpyrene derivative and two cyclodextrins, is highly stable upon irradiation with a UV/vis illuminator. On the other hand, I and my colleagues have developed a variety of fluorescent sensors using pyrene emission switching. One of them is DNA probes in which two pyrenes are connected both at 3' and 5' ends of a single-stranded oligonucleotide. Emission switching occurs from excimer to monomer when the probes hybridized with target DNAs. The DNA probes met antibody structures to produce the following fluorescent sensors. The sensors consist of three functional regions, crown ether (or cyclodextrin), DNA, and pyrene as guest-binding, dimerizing, and sensing sites, respectively. These sensors can detect potassium cation, porphyrin, unsaturated fatty acid by pyrene monomer-excimer switching.

Specific induced circular dichroism and enhanced B to z transitions of duplexes stabilized by chromophore-linked alkynylnucleoside residues.

We have developed an induced circular dichroism (ICD) probe with a chromophore-linked alkynyldeoxyribose skeleton for analyzing higher-order structures of DNA duplexes in the visible-light region. When CG-repeated oligonucleotides (ODNs) with the probe at their 5' ends adopted Z-form duplexes at a high NaCl concentration, strong ICD signals were observed at the absorptive region of the chromophore. On the other hand, their B-form duplexes, formed at a low NaCl concentration, produced a faint ICD signal. The specific ICD for the Z-form duplexes was found to appear only when the chromophores were attached at the 5' ends of each of the ODNs. Furthermore, the chromophoric alkynylnucleoside residues effectively promoted the B to Z transition of the ODN.

Synthesis of versatile fluorescent sensors based on click chemistry: detection of unsaturated fatty acids by their pyrene-emission switching.

Duplex-based, fluorescent sensors bearing cyclodextrins as a guest-binding moiety were synthesised based on Click chemistry, and the sensors selectively detected unsaturated fatty acids by pyrene-emission switching from monomer to excimer.

[DNA duplex-based fluorescence probes/sensors using monomer-excimer switching].

In this review, we report DNA duplex-based fluorescence probes/sensors using pyrene monomer-excimer switching. The review mainly comprises two topics: 1) excimer-monomer switching molecular beacons (EMS-MB) and 2) monomer-excimer switching sensors based on the structural motif of antibodies. The EMS-MBs have two pyrene fluorophores connected both at 3' and 5' ends of a single-stranded oligonucleotide. Emission switching occurs from excimer to monomer accompanying isoemissive points when the probes hybridized with target DNAs. The isoemissive points indicate the presence of only two fluorescent species, nonhybridized and hybridized probes in the mixtures, and thereby unambiguous detection of the targets is available. The probes can detect target 19-mer DNAs and can discriminate the targets from their single-nucleotide mismatches at 1 nM concentration. Furthermore, the EMS-MBs have been recently applied to kinetic study for RNase H activity by Tan et al. The structures and emission-switching properties of the EMS-MBs encourage us to develop a new class of fluorescent sensors based on the structural motif of antibodies. The sensors consist of three functional regions, benzo-15-crown-5 ether (or per-O-methylated beta-cyclodextrin), DNA, and pyrene as guest-binding, dimerizing, and sensing sites, respectively. The crown- and CD-modified sensors can detect potassium cation and porphyrin derivatives, respectively, by monomer-excimer emission switching.

Redox regulation of helical structures in short peptides with an intramolecular ferrocenyl cross-linking agent.

We redox-regulated alpha-helicity of short peptides intramolecularly cross-linked with a ferrocenyl linker between amino acid side chains. The helical content of the cross-linked peptide was estimated to be 56% in the neutral state of the ferrocene core at 25 degrees C. The addition of an oxidant to the solution of the cross-linked peptide enhanced the helicity up to 75%. The increased helical content returned to the same level as that in the previous ferrocene state by further addition of a reductant.

A DNA duplex-based, tailor-made fluorescent sensor for porphyrin derivatives.

We synthesized a DNA-based fluorescent sensor, tailored to the recognition and signaling for hydrophobic guest molecules. The sensor molecule is made of three functional moieties, per-O-methylated beta-cyclodextrin (per-Me-beta-CD), DNA, and pyrene as guest-binding, dimerizing, and signaling ones, respectively. Addition of a porphyrin derivative as a hydrophobic guest molecule induced emission switching of the sensor molecule from monomer to excimer due to the DNA hybridization promoted by the host-guest binding.