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

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 Caenorhabditis elegans. 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. Our tissue-specific knockdown and knockout analyses demonstrated that IIS reduction in neurons and the intestine markedly extended lifespan. DAF-16 activation in neurons resulted in DAF-16 activation in the intestine and vice versa. Our dual gene manipulation method revealed that intestinal and neuronal DAF-16 mediate longevity induced by daf-2 knockout in neurons and the intestine, respectively. In addition, the systemic regulation of intestinal DAF-16 required the IIS pathway in intestinal and neurons. Collectively, these results highlight the importance of the neuronal DAF-16-to-intestinal DAF-16 communication for organismal lifespan regulation.

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 NO3- concentration, and was analyzed in terms of diffusion in the pores of the microparticles and the complex formation of Ln(III). The results indicated that the rate-determining step in Ln(III) extraction was diffusion in the pore solution of the microparticles.

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. Moreover, we found that anti-BGP Q-body with the shortest linker that exhibits the largest antigen-dependency in fluorescence showed the highest binding signal. Similar results were obtained with anti-bisphenol A (BPA) Q-bodies, with inversed correlation with their linker lengths. In the FP assay, when the ATTO 520 labeled Q-body was added with antigen, the Brownian motion of the dye became more active, which resulted in reduced fluorescence anisotropy r. In other words, in the presence of antigen, 1/r showing that the dye mobility is larger than in the absence of its antigen. In addition, anti-BGP Q-body with the largest antigen-dependency in fluorescence showed the highest mobility. Overall, these results clearly suggest that the antigen-dependent fluorescence quenching and recovery of Q-body is caused by the movement of the dye within and around scFv, which moves out of scFv upon binding with its antigen.

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). Following are the steps in the final analytical procedure: (1) 10mg of samples were extracted with 1ml of a 60:40 mixture of methanol and phosphate-buffered saline (PBS); (2) the extract was filtered through a centrifugal 0.2-µm polytetrafluoroethylene membrane filter; (3) the filtrate was diluted 100 times with extraction solvent; (4) 6-µl diluted solution was mixed with 19-µl PBS and 75-µl UQ-body solution; and (5) FIRR was measured under 275-mV excitation light. Herbal cannabis samples containing ≥4.0-mg/g THC gave FIRRs of ≥5.2%. FIRRs of negative samples (cigarette, tea, spice, and so-called "synthetic marijuana") were ≤3.1%. When setting the FIRR threshold to 5.0%, cannabis samples containing ≥4.0-mg/g THC were correctly judged as positive without being affected by false positives caused by the negative samples. This detection limit was lower than total THC level (10-200mg/g) in most herbal cannabis samples seized in Japan. In seven of the 10 cannabis samples, the results of the UQ-body test were comparable with those of the Duquenois-Levine test. Thus, the UQ-body-based immunoassay is presumed to be an effective and objective drug screening method for herbal cannabis; however, to show the true usefulness, it is necessary to test a number of real case samples in the field situation.

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. There were no significant differences in immune parameters, including in antibody responses against the vaccination, between the groups. In the subgroup of the oldest old, defined as ≥85 years of age (n = 27), the antibody responses to the A/H1N1 and B antigens, which were impaired in the placebo group, were improved in the LP group. No significant effects of non-viable L. paracasei MCC1849 were observed in the elderly. A possible beneficial effect in the oldest old should be explored in further large-scale studies.

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 0.0003 and 3 mg L(-1). The coefficients of variation were 7.9% at 0.003 mg L(-1), 5.0% at 0.03 mg L(-1) and 13.7% at 0.3 mg L(-1), respectively. The limit of detection was 0.006 mg L(-1) for DON in wheat. The Q-body showed an antigen-dependent fluorescence enhancement even in the presence of wheat extracts. To validate the analytical method using Q-body, a spike-and-recovery experiment was performed using four spiked wheat samples. The recoveries were in the range of 94.9-100.2%. The concentrations of DON in twenty-one naturally contaminated wheat samples were quantitated by the Q-body method, LC-MS/MS and an immunochromatographic assay kit. The LC-MS/MS analysis showed that the levels of DON contamination in the samples were between 0.001 and 2.68 mg kg(-1). The concentrations of DON quantitated by LC-MS/MS were more strongly correlated with those using the Q-body method (R(2) = 0.9760) than the immunochromatographic assay kit (R(2) = 0.8824). These data indicate that the Q-body system for the determination of DON in wheat samples was successfully developed and Q-body is expected to have a range of applications in the field of food safety.

Ultra Q-bodies: quench-based antibody probes that utilize dye-dye interactions with enhanced antigen-dependent fluorescence.

Recently, we described a novel reagentless fluorescent biosensor strategy named Quenchbody, which functions via the antigen-dependent removal of the quenching effect on a fluorophore that is attached to a single-chain antibody variable region. To explore the practical utility of Quenchbodies, we prepared antibody Fab fragments that were fluorolabeled at either one or two of the N-terminal regions, using a cell-free translation-mediated position-specific protein labeling system. Unexpectedly, the Fab fragment labeled at the heavy chain N-terminal region demonstrated a deeper quenching and antigen-dependent release compared to that observed using scFv. Moreover, when the Fab was fluorolabeled at the two N-termini with either the same dye or with two different dyes, an improved response due to enhanced quenching via dye-dye interactions was observed. On the basis of this approach, several targets, including peptides, proteins, and haptens, as well as narcotics, were quantified with a higher response up to 50-fold. In addition, differentiation of osteosarcoma to osteoblasts was successfully imaged using a similarly fluorolabeled recombinant Fab protein prepared from E. coli. Due to its versatility, this "Ultra-Quenchbody" is expected to exhibit a range of applications from in vitro diagnostics to the live imaging of various targets in situ.

"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. This suggested that the binding of the antigen led to the interruption of a quenching effect caused by the proximity of tryptophan residues to the linker-tagged fluorophore. Using TAMRA-scFv, many targets including peptides, proteins, and haptens including morphine-related drugs could be quantified. Similar or higher sensitivities to those observed in competitive ELISA were obtained, even in human plasma. Because of its versatility, this "quenchbody" is expected to have a range of applications, from in vitro diagnostics, to imaging of various targets in situ.

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. Polarization was increased by adding PPARγ ligands to a solution containing labeled SRC-1 (designated TAMRA-SRC-S) and PPARγ. The disassociation constants (Kd) of the PPARγ synthesized ligands, pioglitazone (221 nM), troglitazone (83.0 nM), and 15-deoxy-Δ12,14-prostaglandin J(2) (15d-ΔPGJ(2)) (156 nM), were determined by this method. Farnesol (2.89 µM) and bixin (21.1 µM), which we have reported to be PPARγ ligands, increased the fluorescent polarization. Their Kd values were in agreement with the ED(50) values obtained in the luciferase assay. The results indicate that the method is valuable for screening natural PPARγ ligands.

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. Here, we developed a method of double incorporation of modified amino acids and fluorescent non-natural amino acids in a quantitative, position-specific manner to obtain modified and fluorescently labeled proteins. To introduce methyllysine, dimethyllysine, trimethyllysine, and acetyllysine, frameshift and amber suppressor tRNAs aminoacylated with modified lysines were synthesized by chemical aminoacylation and supplied to an Escherichia coli cell-free translation system. The immunodetection of the translation products indicated that the modified lysines were incorporated into streptavidin and histone H3 in a quantitative, position-specific manner. Calmodulin derivatives containing a fluorescent non-natural amino acid at the N-terminal region and modified lysines at the Lys115 position were also synthesized, and their binding activity to a calmodulin-binding peptide was analyzed by fluorescence correlation spectroscopy. The results obtained here demonstrate that this method is useful in preparing and analyzing naturally occurring and non-natural modified proteins.

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. TAMRA-linked aminophenylalanine was synthesized and attached to a tRNA having a four-base anticodon, and its incorporation into proteins was examined in an Escherichia coli cell-free translation system. TAMRA-labeled amino acids were successfully incorporated into proteins, although incorporation was allowed only at the N-terminal region. Insertion of two codons encoding a stop codon in the +1 frame before four-base codon suppressed the expression of non-labeled proteins that may have been produced by spontaneous +1 frameshift upstream of the four-base codon. Alternation of the incorporation position affected the expression level of the TAMRA-labeled protein. In addition, alternation of upstream and downstream codons affected the efficiency and accuracy of TAMRA-labeled amino acid incorporation. Based on these results, a novel tag peptide was developed; it contained the four-base codon at the 9th position with optimized upstream and downstream codons. This tag peptide was effective for producing proteins with various fluorescent labels at the N-terminal region.

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. The efficiency of the ester cleavage and the yield of the labeled proteins were dependent on the peptide tag sequence. We demonstrate that the insertion of an asparagine residue between the N-terminal T7 tag and the fluorescent hydroxy acid achieved both quantitative ester cleavage and efficient expression of the labeled proteins. The present method is a potential tool for N-terminal specific labeling of proteins with various compounds.

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. These results indicate that the present method will become a useful tool for the N-terminal specific labeling of proteins.

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. Next, we incorporated BODIPYFL-aminophenylalanine and BODIPY558-aminophenylalanine into different positions of calmodulin as a donor and acceptor pair for fluorescence resonance energy transfer (FRET) using two four-base codons. Fluorescence spectra and polarization measurements revealed that substantial FRET changes upon the binding of calmodulin-binding peptide occurred for the double-labeled calmodulins containing BODIPY558 at the N terminus and BODIPYFL at the Gly40, Phe99 and Leu112 positions. These results demonstrate the usefulness of FRET based on the position-specific double incorporation of fluorescent amino acids for analyzing conformational changes of proteins.

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. These results also indicate that well-designed non-natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system.

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. Transfer RNAs that contained a CCCG anticodon and were aminoacylated with BODIPY FL-labeled amino acids were prepared by a chemical aminoacylation method, and added to an in vitro translation system in the presence of a streptavidin mRNA containing a CGGG codon. SDS-PAGE and Western blot analysis of the synthesized proteins indicate that BODIPY FL-labeled aminophenylalanine derivatives are efficiently incorporated into proteins through the four-base codon decoding.