FRET-Based In-Cell Detection of Highly Selective Supramolecular Complexes of meso-Tetraarylporphyrin with Peptide/BODIPY-Modified Per-O-Methyl-β-Cyclodextrins

Artificial supramolecular systems capable of self-assembly and that precisely function in biological media are in high demand. Herein, we demonstrate a highly specific host-guest-pair system that functions in living cells. A per-O-methyl-β-cyclodextrin derivative (R8-B-CD^Me) bearing both an octaarginine peptide chain and a BODIPY dye was synthesized as a fluorescent intracellular delivery tool. R8-B-CD^Me was efficiently taken up by HeLa cells through both endocytosis and direct transmembrane pathways. R8-B-CD^Me formed a 2 : 1 inclusion complex with tetrakis(4-sulfonatophenyl)porphyrin (TPPS) as a guest molecule in water, from which fluorescence resonance energy transfer (FRET) from R8-B-CD^Me to TPPS was observed. The FRET phenomenon was clearly detected in living cells using confocal microscopy techniques, which revealed that the formed supramolecular R8-B-CD^Me/TPPS complex was maintained within the cells. The R8-B-CD^Me cytotoxicity assay revealed that the addition of TPPS counteracts the strong cytotoxicity (IC₅₀=16 μM) of the CD cavity due to complexation within the cells. A series of experiments demonstrated the bio-orthogonality of the supramolecular per-O-methyl-β-CD/tetraarylporphyrin host-guest pair in living cells.     

Self‐Degradable Lipid‐Like Materials Based on “Hydrolysis accelerated by the intra‐Particle Enrichment of Reactant (HyPER)” for Messenger RNA Delivery

RNA‐based therapeutics is a promising approach for curing intractable diseases by manipulating various cellular functions. For eliciting RNA (i.e., mRNA and siRNA) functions successfully, the RNA in the extracellular space must be protected and it must be delivered to the cytoplasm. In this study, the development of a self‐degradable lipid‐like material that functions to accelerate the collapse of lipid nanoparticles (LNPs) and the release of RNA into cytoplasm is reported. The self‐degradability is based on a unique reaction “Hydrolysis accelerated by intra‐Particle Enrichment of Reactant (HyPER).” In this reaction, a disulfide bond and a phenyl ester are essential structural components: concentrated hydrophobic thiols that are produced by the cleavage of the disulfide bonds in the LNPs drive an intraparticle nucleophilic attack to the phenyl ester linker, which results in further degradation. An oleic acid‐scaffold lipid‐like material that mounts all of these units (ssPalmO‐Phe) shows superior transfection efficiency to nondegradable or conventional materials. The insertion of the aromatic ring is unexpectedly revealed to contribute to the enhancement of endosomal escape. Since the intracellular trafficking is a sequential process that includes cellular uptake, endosomal escape, the release of mRNA, and translation, the improvement in each process synergistically enhances the gene expression.     

Preparation and characterization of the new quaternary chalcogenides Tl-III-IV-S4 (III = Al,Ga, In; IV = Si,Ge)

Five new chalcogenides: TlInSiS₄, TlInGeS₄, TlGaSiS₄, TlGaGeS₄, TlAlGeS₄ were synthesized by the evacuated silica tube method. The crystals are isotypic and belong to the orthorhombic system with space group Pbnm and lattice parameters in the range of, a: 11.53 –11.74, b: 16.50–17.04, c: 7.036–7.353 Å. Infrared and photoacoustic spectra of those compounds are also reported.     

Spatial Distribution of Ions in Groundwater under Agricultural Land

This study investigated the characteristics of shallow groundwater quality under agricultural areas dominated by rice paddy fields, vegetable fields, and a residential zone on an alluvial basin by examining spatial distributions of Cl?, Na+, NO3?, and SO42? concentrations. Seasonal changes in spatial distribution of ion concentrations during one cultivation period and the causes of the changes were discussed. Changes in spatial distribution of Cl? and Na+ concentrations of the groundwater were mainly affected by seasonal changes in precipitation concentrations. NO3? concentration was strongly influenced by fertilization of vegetable fields and a following heavy rainfall. Elevated NO3? concentration was less likely to occur under paddy fields than under vegetable fields. It was suggested that paddy fields might mitigate NO3? contamination of shallow groundwater possibly because of denitrification in paddy soil in an anaerobic state during ponding. As for SO42?, it was acknowledged that there could be a specific point source near a particular observation well.     

The S5-S6 linker of repeat I is a critical determinant of L-type Ca2+ channel conductance

The alpha1-subunits of the skeletal and cardiac L-type calcium channels (L-channels) contain nearly identical pore regions (P-regions) in each of the four internal homology repeats. In spite of this high conservation of the P-regions, native skeletal L-channels exhibit a unitary conductance that is only about half that of native cardiac L-channels. To identify structural determinants of this difference in L-channel conductance, we have characterized unitary activity in cell-attached patches of dysgenic myotubes expressing skeletal, cardiac, and chimeric L-channel alpha1-subunits. Our results demonstrate that the S5-S6 linker of repeat I (IS5-IS6 linker) is a critical determinant of the difference in skeletal and cardiac unitary conductance. The unitary conductances attributable to the wild-type skeletal (CAC6; approximately 14 pS) and cardiac (CARD1; approximately 25 pS) alpha1-subunits expressed in dysgenic myotubes are identical to those observed in native tissues. Chimeric alpha1-subunits containing skeletal sequence for the first internal repeat and all of the putative intracellular loops (SkC15), the IS5-IS6 linker and the intracellular loops (SkC51), or only the IS5-IS6 linker (SkC49) each exhibit a low, skeletal-like unitary conductance (< or = 17 pS). Constructs in which the IS5-IS6 linker is of cardiac origin (CARD1 and CSk9) display cardiac-like conductance (approximately 25 pS). Unitary conductance and the rate of channel activation are apparently independent processes, since both SkC51 and SkC49 exhibit low, skeletal-like conductance and rapid, cardiac-like rates of ensemble activation. These results demonstrate that the IS5-IS6 linker strongly influences the single channel conductance of L-channels in a manner that is independent from the rate of channel activation.     

Respiratory Symptoms and Housing Characteristics

A health and housing questionnaire was administered to children, ages 9-11, living in 24 communities in the United States and Canada. Logistic regression analysis examined the relationship between respiratory health symptoms (bronchitic, asthmatic and lower respiratory) and housing factors. The health risks (expressed as relative odds) were controlled for gender, parental asthma, parental chronic obstructive pulmonary disease and parental education, as well as between-city differences. Lower respiratory symptoms had significantly higher odds ratios reported in older homes (1.12), homes with smokers (1.24), air conditioners (1.14), air cleaners (1.37), and humidifiers (1.47). Home dampness (1.48) and the individual mold and water variables were all significantly associated with increased symptoms. Similar results were reported for bronchitic and asthmatic symptoms. While air conditioners and air cleaners were confounded with symptoms, humidifiers remained significant after controlling for childhood atopy.     

Three dimensional gain analysis of free electron laser with focusing permanent magnet wiggler

A three dimensional simulation software system developed to estimate a free electron laser (FEL) gain has been applied to FEL using a standard plane polarized wiggler and an alternately shifted magnet wiggler. It is seen for the latter wiggler that a large filling factor could be selected and each maximum gain corresponding to each orbit of electron beam concentrates at a certain frequency region of FEL radiation. It is, therefore, implied that a proper shift between the adjacent magnets in the wiggler produces the improvement of the FEL gain.     

Ultrastructure and morphogenesis of human immunodeficiency virus

The ultrastructure and morphogenesis of human immunodeficiency virus (HIV) were elucidated by observation with several techniques including immunoelectron microscopy and cryo-microscopy. The virus particle consists of an envelope, a core and matrix. The virus particles were observed extracellularly as having one of three profiles: (1) a centric or an eccentric electron-dense core, (2) rod-shaped electron-dense core, and (3) doughnut-shaped. HIV-1 particles in the hydrated state were observed by high resolution electron cryo-microscopy to be globular, and the lipid membrane was clearly resolved as a bilayer. Many projections around the circumference were seen to be knob-like. The shapes and sizes of the projections, especially head parts, were found to vary in each projection. By isolation with Nonidet P40 and glutaraldehyde, HIV-1 cores were confirmed to consist of p24 protein by immunogold labeling. When the virus enters the cell, two entry modes were found: membrane fusion and endocytosis. No structures resembling virus particles could be seen in the cytoplasm after viral entry. In HIV-1-infected cells, positive reactions by immuno-labeling suggest that HIV-1 Gag may be produced in membrane-bound structures and transported to the cell surface by cytoskeletons. Then a crescent electron-dense layer was first formed underneath the cell membrane. Finally, the virus particle was released from the cell surface. Several cell clones producing defective particles were isolated from MT-4/HIV-1 cells. Among them, doughnut-shaped or teardrop-shaped particles were seen to be produced in the extracellular space. In the doughnut-shaped particles, Gag p17 and p24 proteins faced each other against the inner electron dense ring, suggesting that the inner ring consists of a precursor Gag protein.     

Synaptic innervation of enkephalinergic neurons by axon terminals immunoreactive to dopamine-beta-hydroxylase in the rat area postrema

A preembedding double immunostaining technique was used to study synaptic relations between enkephalin-like immunoreactive and dopamine-beta-hydroxylase-like immunoreactive neurons in the rat area postrema. Enkephalin-like immunoreactive neuronal perikarya and dendrites were found to receive synapses from dopamine-beta-hydroxylase-like immunoreactive axon terminals. Synapses were also found between the same dopamine-beta-hydroxylase-like immunoreactive neurons. Compared with our previous study, the present results provide morphological evidence that dopaminergic and noradrenergic neurons have different synaptic relations with enkephalinergic neurons, suggesting that physiological functions, especially those related to enkephalinergic neurons, may be different from each other in the area postrema.