Neuronal DAF-16-to-intestinal DAF-16 communication underlies organismal lifespan extension in .

Previous studies have revealed the importance of inter-tissue communications for lifespan regulation. However, the inter-tissue network responsible for lifespan regulation is not well understood, even in a simple organism . To understand the mechanisms underlying systemic lifespan regulation, we focused on lifespan regulation by the insulin/insulin-like growth factor-1 signaling (IIS) pathway; IIS reduction activates the DAF-16/FOXO transcription factor, which results in lifespan extension.

Extraction Mechanism of Lanthanide Ions into Silica-based Microparticles Studied by Single Microparticle Manipulation and Microspectroscopy.

Single porous silica microparticles coated with a styrene-divinylbenzene polymer (SDB) impregnated with octyl(phenyl)-N,N-diisobutylcarbamoylmethylphosphine oxide (CMPO) were injected into an aqueous 3 mol/L nitric acid solution containing trivalent lanthanide (Ln(III)), as a high-level liquid waste model. We used the microcapillary manipulation-injection technique; and the extraction rate of Ln(III), as an Ln(III)-CMPO complex, into the single microparticles was measured by luminescence microspectroscopy. The extraction rate significantly depended on the Ln(III), CMPO, or NO concentration, and was analyzed in terms of diffusion in the pores of the microparticles and the complex formation of Ln(III).

Antigen-responsive fluorescent antibody probes generated by selective N-terminal modification of IgGs.

Novel fluorescent antibody probes, which show antigen-dependent fluorescence responses, were developed by selective N-terminal fluorescent labeling of IgG monoclonal antibodies using a reductive alkylation. Several kinds of IgG against tag peptides and small molecules were successfully utilized to detect antigens in a rapid and quantitative manner.

Construction of dye-stapled Quenchbodies by photochemical crosslinking to antibody nucleotide-binding sites.

We successfully converted an antibody single-chain variable fragment and a full-sized antibody to Quenchbodies, which are a type of powerful fluorescent immunosensor, through ultraviolet-based photochemical crosslinking of an indole-3-butyric acid-conjugated fluorescent dye to the nucleotide-binding sites near the antigen-binding sites.

Insight into the Working Mechanism of Quenchbody: Transition of the Dye around Antibody Variable Region That Fluoresces upon Antigen Binding.

Recently, we reported a novel immunoassay reagent Quenchbody (Q-body): a single chain antibody variable region (scFv) fragment labeled with fluorescent dye, whose fluorescence intensity increases when it binds to the antigen. Here we analyze its working mechanism by immuno- and fluorescence polarization (FP) assays. In an enzyme-linked immunosorbent assay, we found that in the presence of antigen osteocalcin peptide (BGP-C7), more TAMRA-labeled Q-bodies bound to anti-TAMRA antibody than in its absence.

Development of a novel immunoassay for herbal cannabis using a new fluorescent antibody probe, "Ultra Quenchbody".

We developed a novel immunoassay for herbal cannabis based on a new immunoassay principle that uses Ultra Quenchbody ("UQ-body"), a recombinant antibody Fab fragment fluorolabeled at the N-terminal regions. When the antigen binds to anti-Δ(9)-tetrahydrocannabinol (THC) UQ-body, the fluorescence intensity (FI) decreases. The analytical conditions of the immunoassay were optimized based on the FI reduction rate (FIRR).

The effects of non-viable Lactobacillus on immune function in the elderly: a randomised, double-blind, placebo-controlled study.

Forty-two participants in two nursing homes who were ≥65 years of age were randomised to receive a jelly containing 10 billion heat-killed Lactobacillus paracasei MCC1849 cells (LP group) or a placebo jelly without lactobacilli (placebo group) for 6 weeks. Three weeks after beginning jelly intake, all subjects received an influenza vaccination (A/H1N1, A/H2N3 and B). Blood samples were collected before and after the treatment period.

Development of a rapid method for the quantitative determination of deoxynivalenol using Quenchbody.

Quenchbody (Q-body) is a novel fluorescent biosensor based on the antigen-dependent removal of a quenching effect on a fluorophore attached to antibody domains. In order to develop a method using Q-body for the quantitative determination of deoxynivalenol (DON), a trichothecene mycotoxin produced by some Fusarium species, anti-DON Q-body was synthesized from the sequence information of a monoclonal antibody specific to DON. When the purified anti-DON Q-body was mixed with DON, a dose-dependent increase in the fluorescence intensity was observed and the detection range was between 0.

"Quenchbodies": quench-based antibody probes that show antigen-dependent fluorescence.

Here, we describe a novel reagentless fluorescent biosensor strategy based on the antigen-dependent removal of a quenching effect on a fluorophore attached to antibody domains. Using a cell-free translation-mediated position-specific protein labeling system, we found that an antibody single chain variable region (scFv) that had been fluorolabeled at the N-terminal region showed a significant antigen-dependent fluorescence enhancement. Investigation of the enhancement mechanism by mutagenesis of the carboxytetramethylrhodamine (TAMRA)-labeled anti-osteocalcin scFv showed that antigen-dependency was dependent on semiconserved tryptophan residues near the V(H)/V(L) interface.

Development of a novel PPARγ ligand screening system using pinpoint fluorescence-probed protein.

The activation of peroxisome-proliferator-activated receptor-γ (PPARγ), which plays a central role in adipocyte differentiation, depends on ligand-dependent co-activator recruitment. In this study, we developed a novel method of PPARγ ligand screening by measuring the increase in fluorescent polarization accompanied by the interaction of a fluorescent co-activator and PPARγ. Sterol receptor co-activator-1 (SRC-1), a major PPARγ co-activator, was probed by fluorescent TAMRA by the Amber codon fluorescence probe method.

Biosynthesis of proteins containing modified lysines and fluorescent labels using non-natural amino acid mutagenesis.

The preparation of posttranslationally modified proteins is required to investigate the function and structure of modified proteins. However, homogeneously modified proteins are not easily isolated from natural sources or prepared using modification enzymes. Non-natural amino acid mutagenesis has enabled us to incorporate modified amino acids into specific positions of proteins in both cell-free and in-cell translation systems using tRNAs that are aminoacylated with modified amino acids.

Incorporation of fluorescent non-natural amino acids into N-terminal tag of proteins in cell-free translation and its dependence on position and neighboring codons.

Fluorescence labeling is a useful technique for structural and functional analyses of proteins. In a previous study, we developed position-specific incorporation of visible wavelength fluorescent non-natural amino acids carrying relatively small BODIPY fluorophores into proteins, in response to a four-base codon CGGG. Here, we have expanded this position-specific fluorescence labeling method to include relatively large non-natural amino acids carrying photostable rhodamine dyes.

N-terminal specific fluorescence labeling of proteins through incorporation of fluorescent hydroxy acid and subsequent ester cleavage.

We have developed a novel method to attach a fluorescent label at the N terminus of proteins through a four-base codon-mediated incorporation of a fluorescent hydroxy acid and subsequent cleavage of the ester bond in a cell-free translation system. We found that a fluorescent-labeled p-amino-L-phenyllactic acid was successfully incorporated downstream of N-terminal tag peptides in response to a CGGG codon, and the tag peptides could be removed through ester cleavage to leave the fluorescent hydroxy acid at the N terminus of the proteins. Immunoprecipitation analysis revealed that ester cleavage occurred spontaneously during the translation reaction.

N-terminal specific fluorescence labeling of proteins through fourbase codon-mediated incorporation of fluorescent hydroxy acid.

Fluorescence labeling of proteins is a useful tool for protein structural and functional analysis. We developed here a novel method to attach a fluorescence labeling at the N terminus of proteins through the incorporation of a fluorescent hydroxy acid and subsequent hydrolysis of the ester bond in a cell-free translation system. We found that N-terminal tagged proteins containing p-(BODIPYFL-amino)-L-phenyllactic acid at the downstream of the tag peptides were efficiently synthesized and the resulting ester bonds were hydrolyzed during the translation reaction.

FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids.

We designed and synthesized new, fluorescent, non-natural amino acids that emit fluorescence of wavelengths longer than 500 nm and are accepted by an Escherichia coli cell-free translation system. We synthesized p-aminophenylalanine derivatives linked with BODIPY fluorophores at the p-amino group and introduced them into streptavidin using the four-base codon CGGG in a cell-free translation system. Practically, the incorporation efficiency was high enough for BODIPYFL, BODIPY558 and BODIPY576.

Position-specific incorporation of dansylated non-natural amino acids into streptavidin by using a four-base codon.

Novel non-natural amino acids carrying a dansyl fluorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG four-base codon in an Escherichia coli in vitro translation system. 2,6-Dansyl-aminophenylalanine (2,6-dnsAF) was found to be incorporated into the protein more efficiently than 1,5-dansyl-lysine, 2,6-dansyl-lysine, and 1,5-dansyl-aminophenylalanine. Fluorescence measurements indicate that the position-specific incorporation of the 2,6-dnsAF is a useful technique to probe protein structures.

Incorporation of fluorescently labeled nonnatural amino acids into proteins in an E. coli in vitro translation system.

Various nonnatural amino acids has been incorporated into proteins by using four-base codons in an E. coli in vitro translation system. Here, design and synthesis of novel fluorescently labeled nonnatural amino acids and their incorporation into proteins were investigated.