Structural and functional glycosphingolipidomics by glycoblotting with aminooxy-functionalized gold nanoparticle

Glycosphingolipids (GSLs) synthesized in Golgi apparatus by sequential transfer of sugar residues to a ceramide lipid anchor are ubiquitously distributing on vertebrate plasma membranes. Standardized method allowing for high throughput structural profiling and functional characterization of living cell surface GSLs is of growing importance because they function as crucial signal transduction molecules in various processes of dynamic cellular recognitions. However, methods are not available for amplification of GSLs, while the genomic scale PCR amplification permits large-scale mammalian proteomic analysis.　Here we communicate such an approach to a novel "omics", namely glycosphingolipidomics based on the glycoblotting method. The method, which involves selective ozonolysis of the C-C double bond in ceramide moiety and subsequent enrichment of generated GSL-aldehydes by chemical ligation using aminooxy-functionalized gold nanoparticle (aoGNP) should be of widespread utility for identifying and characterizing whole GSLs present in the living cell surfaces. The present protocol using glycoblotting permitted MALDI-TOFMS-based high throughput structural profiling of mouse brain gangliosides such as GM1, GD1a/GD1b, and GT1b for adult or GD3 in case for embryonic mouse. When mouse melanoma B16 cells were subjected to this protocol, it was demonstrated that gangliosides enriched from the plasma membranes are only GM3 bearing microheteogeneity in the structure of N-acyl chain. Surface plasmon resonance analysis revealed that aoGNP displaying whole GSLs blotted from mouse B16 melanoma cell surfaces can be used directly for monitoring specific interaction with self-assembled monolayer (SAM) of Gg3Cer (gangliotriaosylceramide). Our results indicate that GSL-selective enrichment onto aoGNP from living cell surfaces allows for rapid reconstruction of plasma membrane models mimicking intact GSL-microdomain feasible for further structural and functional characterization

Perceptual Continuity and Naturalness of Expressive Strength in Singing Voices Based on Speech Morphing

This paper experimentally shows the importance of perceptual continuity of the expressive strength in vocal timbre for natural change in vocal expression. In order to synthesize various and continuous expressive strengths with vocal timbre, we investigated gradually changing expressions by applying the STRAIGHT speech morphing algorithm to singing voices. Here, a singing voice without expression is used as the base of morphing, and singing voices with three different expressions are used as the target. Through statistical analyses of perceptual evaluations, we confirmed that the proposed morphing algorithm provides perceptual continuity of vocal timbre. Our results showed the following: (i) gradual strengths in absolute evaluations, and (ii) a perceptually linear strength provided by the calculation of corrected intervals of the morph ratio by the inverse (reciprocal) function of an equation that approximates the perceptual strength. Finally, we concluded that applying continuity was highly effective for achieving perceptual naturalness, judging from the results showing that (iii) our gradual transformation method can perform well for perceived naturalness

Effects of Sulfur Amino Acids on Tyrosyl or Serine/Threonine Phosphorylation and Translocation of Cytosolic Compounds to Cell Membrane in Stimulus-treated Human Neutrophils

We investigated the effects of various sulfur amino acids on the phosphorylation of proteins and the translocation of cytosolic compounds to cell membrane in stimulus-treated human neutrophils using specific monoclonal antibodies. D,L-homocysteine and D,L-homocysteine-thiolactone enhanced fMLP-induced tyrosyl phosphorylation of proteins and the translocation of p47phox, p67phox, and rac to the cell membrane in a concentration-dependent manner. L-cystathionine, NAc-L-cysteine and carboxymethylcysteine suppressed the tyrosyl phophorylation and translocation of cytosolic compounds to the cell membrane. L-cystathionine, L-cysteine and NAc-L-cysteine suppressed PMA-induced serine/threonine phosphorylation and the translocation of cytosolic compounds to the cell membrane. L-cysteine, NAc-L-cysteine and D,L-homocysteine enhanced AA-induced serine/threonine phosphorylation and the translocation of cytosolic compounds to the cell membrane, but L-cystathionine had opposite effects. These results indicated that the effects of sulfur amino acids on tyrosyl or serine/threonine phosphorylation and the translocation of p47phox, p67phox, and rac to the cell membrane in the stimulus-treated human neutrophils were in parallel with those of the stimulus-induced superoxide generation reported in previous paper. L-cysteine, D,L-homocysteine and L-cystathionine weakly inhibited lipid peroxidation, but the other sulfur amino acids tested had no effect.</p

To Be Supported, or Not to Be: Images of Older People in Policy and the Reality in Local Communities in Japan

Social innovation is not only about tangible new products, services, policies, and laws, but also about changes in societal perceptions, values, and norms. In Japan, current policies for older people, including Long-Term Care Insurance, tend to focus on medical and long-term care and other forms of “support” for older adults such as a pension. Naturally, these policies depict older adults as the “beneficiaries,” or the ones in need of support. However, when we look back at pre-modern Japan, it was not always like that. Although older adults did depend on support from family and community members, they also played an active role as a laborer and caretaker as well as providing useful knowledge for their family and community. Moreover, currently, in different areas suffering from a sharp decline in population, such as Okayama prefecture in western Japan, older people are actually playing the role of the supporter for groups of people who are in need, not only the aged population but also other demographics including young children and parents. Based on this historic “tradition” and the present reality, this paper argues that we need to reestablish the image of (at least some) older people as capable of taking a more active and responsible role in society, and position them as such in the new “welfare society” systems in order to replace the conventional “welfare state” model

Design and synthesis of a new chromophore, 2-(4-nitrophenyl)benzofuran, for two-photon uncaging using near-IR light

C. K. acknowledges the HPC resources of CINES and of IDRIS under the allocations 2014-[x2014080649] made by GENCI (Grand Equipement National de Calcul Intensif).International audienceA new chromophore, 2-(4-nitrophenyl)benzofuran (NPBF), was designed for two-photon (TP) uncaging using near-IR light. The TP absorption (TPA) cross-sections of the newly designed NPBF chromophore were determined to be 18 GM at 720 nm and 54 GM at 740 nm in DMSO. The TP uncaging reaction of a caged benzoate with the NPBF chromophore quantitatively produced benzoic acid with an efficiency (δu) of ∼5.0 GM at 740 nm. The TP fragmentation of an EGTA unit was observed with δu = 16 GM. This behavior makes the new chromophore a promising TP photoremovable protecting group for physiological studies

高速液体クロマトグラフィーによる食品中の農薬分析

Reversed-phase high performance liquid chromatography (HPLC) using acetonitrile-water as a mobile phase and UV detection is performed for a rapid and simultaneous determination of 25 pesticides of different types, namely organophosphorus, organochlorine, carbamates, triazines and nitroaniline in fruits, vegetables and tea leaves. The pesticides were extracted with acetone and then partitioned with dichloromethane. Clean-up of the samples was accomplished by open-column liquid chromatography with florisil. Eleven pesticides were detected from 22 among 35 samples.The detection limits of the present method were less than 0. 001μg/g

1997年岡山県下に発生した集団食中毒患者から分離された腸管出血性大腸菌EHEC O157 : H7のパルスフィールドゲル電気泳動法による遺伝子解析

Thirteen enterohemorrhagic Escherichia coli O157 : H7 (EHEC) isolates derived from the patients of an outbreak in the R-hospital in Okayama Japan and one isolated from the ingredients of Japanese noodles in June 1997 were analyzed by molecular typing using pulsed-field gel electrophoresis (PFGE). The PFGE patterns of the patients were almost the same as the patterns of the Japanese noodle ingredients. Therefore, the EHEC O157 : H7 derived from the food was considered to have caused the outbreak in the R-hospital. The molecular typing of isolates from the patients and the Japanese noodle ingredients was almost the same as that of isolates from outbreaks in Hiroshima and Fukuoka prefectures classified as type Ia in 1996 by PFGE analysis. These results indicate that EHEC O157 : H7 strains with a similar PFGE type Ia pattern have already spread throughout western Japan since last year.1997年6月に岡山県下のR-病院で腸管出血性大腸菌EHEC O157 : H7による集団食中毒が発生した｡その患者および食材である日本そばから分離されたEHEC O157 : H7の菌株をパルスフィールドゲル電気泳動法(PFGE)を使用して遺伝子解析を行った｡13名の患者から分離された菌株の遺伝子型は日本 そばから分離された菌株と一致した｡従って日本そばから分散されたEHEC O157 : H7が集団食中毒の原因であることが解った｡さらにこれらの菌株の遺伝子型は1996年に広島県および福岡県の西日本に集団食中毒が発生した菌株のタイプIa型と類似していた｡このことはこの遺伝子タイプの類似した菌株の感染が西日本で去年から広がっていることを示している