Search

This study demonstrates bioluminescent indicators for determining stress hormones in mammalian cells. A genetically encoded bioluminescent probe for stress sensing was first synthesized with a LXXLL, motif-linked ligand binding domain of the glucocorticoid receptor (GR LBD), which was then sandwiched between the fragments of Gaussia luciferase (GLuc). This prototype of the bioluminescent indicators was carefully modified with a circular permutation (CP) and/or a corepressor motif. The first notable appearance by cofusion of a corepressor motif to the probe was the biphasic dose-response curves of the indicator to cortisol. A CP largely improved the detection limit of the indicator to cortisol up to 100 times. Fabrication of both CP and the attachment of a corepressor motif in the indicator synergistically contributes to (i) the lower detection limit and wider dynamic range and (ii) the enhanced absolute luminescence and ligand selectivity. This study is the first example that contribution of core-pressor motifs to single-chain probes was investigated. This study also provides new insight into improving the sensorial properties of single-chain probes with CP.

The goal of the present study is to develop a small luminescence template, in which any protein may be incorporated, for illuminating the dynamics of protein signaling. We first determined optimal fragmentation sites of Gaussia princeps-derived luciferase (GLuc). The utility of the template was demonstrated with calmodulin (CAM) and a peptide-linked CAM, which were sandwiched between the fragments of split GLuc dissected at Q105. Living mammalian cells with the probe quickly emitted luminescence in response to endo- and exogenous [Ca.sup.2+] levels. The applicability of the template was expanded to the visualization of the phosphorylation of the estrogen receptor and interaction of the androgen receptor with a peptide via an intramolecular complementation between GLuc fragments. It also provides the smallest bioluminescence template ever synthesized. Considering that conformational changes of proteins generally occur for signal transductions, the present luminescence template will contribute to the exploration of intracellular molecular events with signaling proteins.

A new method for phylogenetic classification of bacterial swains using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is proposed. This method was developed using a bioinformatics-based approach to the rapid identification of bacteria as previously proposed by Demirev and co-workers, which uses ribosomal proteins composed of ~50 subunit proteins as biomarkers. Although the amino acid sequences of ribosomal proteins are highly conserved, slight sequence variations can occur at the strain level. Since ribosomal subunit proteins are a complex of housekeeping proteins that have different phylogenetic evolution rates, sequence variation detected as mass differences by MALDI-MS may be useful for the phylogenetic classification of bacteria at strain level. In our proposed method, the first step is the selection of reliable biomarkers through characterization of the expressed ribosomal subunit proteins of a reference strain (usually a genome-sequenced strain) by MAIDI-MS. The observed masses in the MALDI mass spectra of cell lysates of sample strains are then compared with the biomarker masses of the reference swain. The biomarkers for each sample strain were designated as present or absent at the reference masses, indicated by 1 or 0, respectively, which were summarized in a table. This table is processed by cluster analysis, generating a phylogenetic tree. In this study, the success of this approach was confirmed by classification of Pseudomonas putida strains because its classification is much more complicated than that of other bacterial strains. Forty-three reliable biomarkers were selected from ribosomal subunit proteins of a genome-sequenced strain, P. putida KT2440. The numbers and kinds of biomarkers observed for 16 strains of P. putida, including different biovars, were markedly different, reflecting the variety of the strains. The classification results by the proposed method were highly comparable to those based on the DNA gyrase subunit B gene (gyrB) sequence analysis, suggesting our proposed method would be a useful high-throughput method for phylogenetic classification of newly isolated bacteria.

A novel ionization platform for matrix-free laser desorption/ionization-mass spectrometry (LDI-MS) was developed using self-assembled germanium nanodots (GeNDs) of uniform size (~150-200-nm width and ~50-nm height) grown on a silicon wafer produced by molecular beam epitaxy. The performance of LDI-MS using GeNDs (GeND-MS) was investigated through measurements of a broad range of analytes, including peptides, proteins, synthetic oligomers, and polymer additives. Mass spectra of tryptic digests were clearly observed even for the mass range lower than m/z 800 without obstructive peaks. A detection limit of subfemtomole level was achieved for angiotensin-I. The upper limit of detectable mass range was ~17 kDa (myoglobin). GeND-MS also has potential for application to the characterization of industrial compounds. Almost accurate molecular weight distribution was obtained for a nonionic surfactant (Triton X-100) and for poly(ethylene glycol) oligomer. Furthermore, a brominated flame retardant, tetrabromobisphenol-A bis(2,3-dibromopropyl ether), was successfully ionized with less fragmentation, a result not obtainable by matrix-assisted laser desorption/ionization-mass spectrometry or desorption/ionization on porous silicon-mass spectrometry.

Click beetle luciferase (CBLuc) is insensitive to pH, temperature, and heavy metals, and emits a stable, highly tissue-transparent red light with luciferin in physiological circumstances. Thus, the luminescence signal is optimal for a bioanalytical index reporting the magnitude of a signal transduction of interest. Here, we validated a single-molecule-format complementation system of split CBLuc to study signal-controlled protein--protein (peptide) interactions. First, we generated 10 pairs of N- and C-terminal fragments of CBLuc to examine respectively whether a significant recovery of the activity occurs through the intramolecular complementation. The ligand binding domain of androgen receptor (AR LBD) was connected to a functional peptide sequence through a flexible linker. The fusion protein was then sandwiched between the dissected N- and C-terminal fragments of CBLuc. Androgen induces the association between AR LBD and a functional peptide and the subsequent complementation of N- and C-terminal fragments of split CBLuc inside the single-molecule-format probe, which restores the activities of CBLuc. The examination about the dissection sites of CBLuc revealed that the dissection positions next to the amino acids D412 and I439 admit a stable recovery of CBLuc activity through an intramolecular complementation. The ligand sensitivity and kinetics of the single molecular probe with split CBLuc were discussed in various cell lines and in different proteinpeptide binding models. The probe is applicable to developing biotherapeutic agents on the AR signaling and for screening adverse chemicals that possibly influence the signal transduction of proteins in living cells or animals.

A novel postcolumn photooxidation reactor with remarkably high efficiency was developed. It was installed into a system of liquid chromatography (LC) combined with inductively coupled plasma mass spectrometry (ICPMS) using a hydride generation (HG) technique to enable sensitive speciation of organic arsenicals. An LC eluent was passed into a reaction tube that runs through a low-pressure mercury lamp of a high-efficiency photooxidation (HEPO) reactor. It provided efficient irradiation with vacuum ultraviolet light at 185 nm, which induced oxidative decomposition of persistent organic arsenicals, for example, arsenobetaine, to the final product, arsenate, for an extremely short time, 3.5 s, without using any oxidizing agents. That arsenate was subsequently detectable by HG-ICPMS. In contrast, conventional photooxidation reactors require more than 100 s for completion, even when using a peroxodisulfate solution as an oxidizing agent. Because of the absence of broadening and dilution of the LC-separated peaks caused by mixing with the oxidant solution, the detection limits for 10 arsenic species including the persistent organic arsenicals by the proposed LC-HEPO-HG-ICPMS ranged from 2.3 to 18 ng of As/L, which were about one-tenth as low as those by LC-ICPMS. The proposed method revealed for the first time the occurrence of 20 arsenic species, including the unknown trace species, which were undetectable by LC-ICPMS and LC-HG-ICPMS, in human urine.

A sensitive method for the determination of ultratrace organotin species in seawater is described. The merits and demerits of derivatization methods using Grignard reagent or sodium tetracethylborate (NaBEt(sub 4) were evaluated in terms of derivatigation efficiency, applicability to the programmed temperature vaporization (PTV) method, and procedural blanks. The sensitivity of the gas chromatography/inductively coupled plasma mass spectrometry (GC/ICPMS) was improved by more than 100-fold by operating the shield torch at normal plasma conditions, compared with that obtained without using it. The absolute detection limit as tin reached subfemtogram (fg) levels. Furthermore, the detection limit in terms of relative concentration was improved 100-fold by using the PTV method, which enabled the injection of a large sample volume of as much as 100 mu L without loss of analyte. When the organotin species in seawater were extracted into hexane with a preconcentration factor of 1000 after ethylation with NaBEt(sub 4) and 100 mu L aliquot of the extract was injected into the GC, the instrumental detection limit in relative concentration reached 0.01 pg/L in original seawater. Sources of contamination of organotin species during the sample preparation were examined, and a purification method of NaBEt(sub 4) was developed. Finally, the method was successfully applied to open ocean seawater samples containing organotin species at the level of 1-100 pg/L.

Partitioning behaviors of analyte cations and anions in 'electrostatic ion chromatography (electrostatic IC)' were investigated. Detectors of ICP-AES (inductively coupled plasma atomic emission spectrometry), UV absorption, and conductivity were connected on-line to the electrostatic IC and used for the selective detection of cations, anions, and 'ion-pairing-like forms' (both cations and anions), respectively. When a mixed electrolytic aqueous solution containing cations A, C, ..., Y, and anions B, D, ..., Z, was passed through the electrostatic IC column, the analyte ions, which dissociated from the original salts AB, CD, ..., and YZ, were each redistributed and forced into a new state. This new state was termed an ion-pairing-like form because of the simultaneous electrostatic attraction and repulsion interactions. The same species of anion or cation are found in more than one ion-pairing-like form; however, they occur in different volumes. What is more, each ion-pairing-like form has a different priority of formation. Born's equation was used to evaluate that priority. Retention time of the ion-pairing-like form was dependent on the species of the anion and the charge of the cation. All ion-pairing-like forms can be simultaneously separated with the exception of those having the same anion species as well as the same cation charge. Ion chromatography using a strong/strong positive/negative charged zwitterionic stationary phase with pure water as the mobile phase was termed electrostatic ion chromatography.

Investigates partitioning behaviors of analyte cations and anions in electrostatic ion chromatography. Ion redistribution; Priority among the ion-pairing-like forms.

In this study, a rational synthesis of superluminescent variants from marine luciferases with prolonged bioluminescence has been demonstrated. A putative active site of a model marine luciferase, Gaussia princeps Luciferase (GLuc), was assigned and modified by a site-directed mutagenesis. The potent variants were found to generate up to 10 times stronger bioluminescence, emitting red shifts of up to 33 nm with natural coelenterazine than native GLuc, rendering an efficient optical signature in bioassays. The advantageous properties were demonstrated with mammalian two-hybrid assays, single-chain probes, and metastases of murine B16 melanoma in BALB/c nude mice. The unique ideas for engineering GLuc are proved to be valid even for other marine luciferases.

This study demonstrates a unique, nontranscriptional assay system based on molecular tension of a luciferase artificially appended by protein-protein binding. We hypothesized that an artificially appended molecular tension to a full-length luciferase may diversify the enzymatic activity through a modification of the active site. For the basic probe design, a full-length luciferase was sandwiched between two component proteins of interest. The length of flexible linkers between the components was minimized to exert an efficient molecular tension to the sandwiched luciferase. When N- and C-terminal ends of Renilla luciferase 8 were flanked by the ligand-binding domain of human estrogen receptor alpha (ER LBD) and SH2 domain of Src, named ERS, this simple probe was surprisingly sensitive to estrogens. The luminescence spectra by ERS were largely enhanced by an addition of 4-hydroxytamoxifen (OHT), 17beta-estradiol, and genistein. The detection limit of ERS reached 1 nM OHT. Quantum yield (QY) and Michaelis-Menten constant of ERS were found to be 6.3% and 94.3 muM, respectively. The enzymatic activities of ERS are also governed by different types of coelenterazine (CTZ) variants. The two hydroxy groups in CTZ are critical for the enzymatic activities of ERS. This study is the first example that an artificially appended molecular tension to a full-length luciferase can be taken as an optical signature upon molecular imaging. This study also provides new insight into the construction of a new lineage of bioluminescent probes for estimating protein-protein interactions.

Engineered bioluminescent enzymes provide a creative approach for illuminating the intracellular molecular events. We demonstrate a new strategy for molecular imaging of bioactive small molecules using circularly permutated luciferases (cpLuc), derived from Gaussia princeps (GLuc), Photinus pyralis (FLuc), and Pyrearinus termitilluminans (CBLuc): (i) the luciferases were first dissected into two fragments, (ii) the original N- and C-termini were linked with a GS linker and the new termini were created in an appropriate site, and (iii) the new ends were correspondingly linked with proteins of interest, e.g., a ligand-binding domain (LBD) and an LBD-recognition protein. When the ends of the cpCBLuc were linked with LBD of estrogen receptor (ER) and Src homology 2 domain of Src (SH2 of Src), the estrogen can trigger an intramolecular ER LBD-Src SH2 binding. This assembly subsequently provokes an approximation of the adjacent fragments of cpCBLuc recovering the enzyme activity. These probes were surprisingly stable in the mammalian cells and largely exhibited a decreased background luminescence (e.g., 1000 times in case of cpGLuc) and improved signal-to-noise ratios, compared with the non-CP indicators. This study offers a new strategy for luciferase-aided probing systems. Our study is the first to fabricate circular permutation (CP) in luciferases for tracing the molecular dynamics of proteins.

Bioactive small molecules, including steroids, activate multiple signaling pathways in mammalian cells. However, current technologies cannot illuminate such multiple effects of a ligand in mammalian cells. Here, we demonstrate integrated-molecule-format multicolor systems simultaneously visualizing bifacial activities of a ligand, where estrogen receptor alpha (ERalpha) was exemplified to demonstrate the present technology. First, we developed a single-molecule-format probe emitting red bioluminescence for imaging interaction between the phosphorylated ligand binding domain of ERalpha (ER LBD) and the Src homology-2 (SH2) domain of Src. The SH2 domain-linked ER LBD was sandwiched between dissected N- and C-terminal fragments of Pyrophorus plagiophthalamus (click beetle) luciferase emitting red bioluminescence. Second, another single-molecule-format bio-luminescent probe emitting green bioluminescence was constructed to visualize intramolecular interaction between ER LBD and LXXLL motifs. Mammalian cells carrying the two probes emit red and/or green light in response to agonistic and antagonistic activities of a ligand, which correspond to its genomic and nongenomic activities, respectively. Third, the two probes were assembled to make an single-molecule-format multicolor indicator, in which all of the components for ligand sensing and multiple-light emission were integrated. The probe emitted characteristic light spectra in response to various agonists and antagonists. This is the first example where (i) protein phosphorylation was recognized with a single bioluminescent probe and (ii) bifacial activities of a ligand, either agonistic or antagonistic, were simultaneously visualized with multiple colors.

Rapid identification of bacteria by a bioinformatics-based approach, which processes the mass spectra observed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), relies on the calculated masses of ribosomal subunit proteins as biomarkers predicted from amino acid sequences found in protein sequence databases. To verify the actual state of the registered sequence information, a simple intact protein analysis by MALDI-MS using cell lysates as samples was applied to the characterization of ribosomal proteins from genome-sequenced Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus strains. This method avoided the risk of loss of some subunit proteins and the formation of disulfide bonds during the purification of ribosomal proteins. By comparing this with the MALDI mass spectra of different strains and carrying out manual inspection of sequence information, a total of five errors in N-terminal amino acid sequences were identified. After sequence correction, approximately 40 out of 53 subunit proteins could be assigned, considering N-terminal methionine loss only as a post-translational modification. These show promise for use as practical biomarkers for the rapid identification of S. thermophilus and L. bulgaricus. After verification of these amino acid sequences, mass differences relative to those of genome-sequenced strains have the potential for distinguishing bacteria at the strain level.

Androgen receptor (AR) induces cell proliferation by increasing the kinase activity of Src. We describe an approach for discriminating agonist and antagonist in a nongenomic steroid-signaling pathway using an association of AR with Src. We constructed a pair of genetically encoded indicators, where N- and C-terminal fragments of split firefly luciferase (FLuc) were fused to AR and Src, respectively. The fusion proteins with AR and Src are localized in the cytoplasm and on the plasma membrane, respectively. Upon being activated with androgen, AR undergoes an intramolecular conformational change and binds with Src. The association causes the complementation of the split FLuc and recovery of FLuc activity. The resulting luminescence intensities were taken as a measure of the rapid hormonal activity of steroids in the nongenomic AR signaling. Ten minutes are required for the AR-Src association by 5alpha-dihydroxytestosterone (DHT), which was completely inhibited by an antagonist, cyproterone acetate. The activities of ligands in the nongenomic pathway of AR were compared with those in the genomic pathway obtained on the basis of the nuclear trafficking of AR in mammalian cells. The comparison revealed that DHT and testosterone activate both genomic and nongenomic pathways of AR. 17beta-Estradiol and progesterone were found to be specific activators only for the genomic signaling pathway of AR. On the other hand, procymidone exhibited a specific activity only for the nongenomic signaling pathway of AR. The present approach is the first example addressing the agonistic and antagonistic activities of ligands in a nongenomic pathway of AR.

Ligand-induced conformational changes of nuclear hormone receptors (NRs) are an important start-up of various hormone signaling. However, little is known of the bioanalytical use of the hormone-induced conformational changes of NRs. Here, we describe a generally applicable bioluminescence assay with a genetically encoded bioluminescent indicator to determine androgenicity of ligands based on the intramolecular association of the ligand binding domain of androgen receptor (AR LBD) with the "FQNLF" motif in the N-terminal domain of AR (AR NTD). Firefly luciferase (FLuc) was dissected into N-terminal (1-415 AA) and C-terminal (416-550 AA) fragments. The AR LBD and FQNLF motif of AR NTD were sandwiched between the dissected fragments of FLuc to construct a single molecule-format bioluminescent probe. Androgens induce the association of AR LBD with the FQNLF motif in the NTD, and the subsequent complementation of N- and C-terminal fragments of FLuc partially restores the activities of FLuc. A 10-5 M solution of 5alpha-dihydroxytestosterone (DHT) induced a quick increase in the luminescence intensities from cervical carcinoma-derived HeLa cells carrying the genetic indicator, which reached a plateau in 9 min, whereas DHT withdrawal from the cells by a medium change decreased the luminescence with a slower time course, i.e., approximately 2 h until returning to the background luminescence. The present luminescent indicator was found to exhibit high agonist selectivity and reproducible recovery of the luminescence to a repeated androgen addition and withdrawal. This is the first contribution that cellular signaling steps can be imaged with bioluminescence using a single molecule-format bioluminescence probe (Simbi), in which all the components required for a signal sensing and visualization are integrated. Simbi is applicable to developing biotherapeutic agents effecting to the AR signaling, and for screening adverse chemicals that possibly influence the signal transduction of AR.