[Show abstract][Hide abstract] ABSTRACT: Biosensors using live cells are analytical devices that have the advantage of being highly sensitive for their targets. Although attention has primarily focused on reporter gene assays using functional promoters, cell viability assays are still efficient. We focus on long non-coding RNAs (lncRNAs) that are involved in the molecular mechanisms associated with responses to cellular stresses as a new biological material. Here we have developed human live cells transfected with lncRNAs that can be used as an intelligent sensor of cytotoxicity for a broad range of environmental stresses. We identified three lncRNAs (GAS5, IDI2-AS1, and SNHG15) that responded to cycloheximide in HEK293 cells. Overexpression of these lncRNAs sensitized human cells to cell death in response to various stresses (cycloheximide, ultraviolet irradiation, mercury II chloride, or hydrogen peroxide). In particular, dual lncRNA (GAS5 plus IDI2-AS1, or GAS5 plus SNHG15) overexpression sensitized cells to cell death by more cellular stresses. We propose a method for highly sensitive biosensors using overexpression of lncRNAs that can potentially measure the cytotoxicity signals of various environmental stresses.
Journal of Bioscience and Bioengineering 11/2014; 119(5). DOI:10.1016/j.jbiosc.2014.10.012 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this study, we focused on two biological products as ideal tools for toxicological assessment: long non-coding RNAs (lncRNAs) and human-induced pluripotent stem cells (hiPSCs). lncRNAs are an important class of pervasive non-protein-coding transcripts involved in the molecular mechanisms associated with responses to cellular stresses. hiPSCs possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical issues associated with human embryonic stem cells. Here, we identified six novel lncRNAs (CDKN2B-AS1, MIR22HG, GABPB1-AS1, FLJ33630, LINC00152, and LINC0541471_v2) that respond to model chemical stresses (cycloheximide, hydrogen peroxide, cadmium, or arsenic) in hiPSCs. Our results indicated that the lncRNAs responded to general and specific chemical stresses. Compared with typical mRNAs such as p53-related mRNAs, the lncRNAs highly and rapidly responded to chemical stresses. We propose that these lncRNAs have the potential to be surrogate indicators of chemical stress responses in hiPSCs.
PLoS ONE 08/2014; 9(8):e106282. DOI:10.1371/journal.pone.0106282 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An exchange between lanthanide ions (Ln3+) in a solution and coordinated yttrium ions (Y3+) takes place in a coordination polymer (CP) formed by Y3+ and di-(2-ethylhexyl) phosphoric acid (Hdehp). Through this cation exchange, Ln3+ is adsorbed on the CP depending on the coordination power with Hdehp. Accordingly, the Ln3+ with the larger atomic number is more preferably adsorbed into the CP. This adsorption is affected by the concentration of H+, CH+, in the solution. For example, at CH+ = 10-5 M, Y3+ is replaced directly by the incoming Ln 3+ with a 1:1 stoichiometry. In a limited higher CH+ region, the CP becomes a gel and the Ln3+/Y3+ exchange is enhanced therein.
Solvent Extraction Research and Development Japan 01/2014; 21(1):83-87. DOI:10.15261/serdj.21.83 · 1.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study demonstrates the creation of artificial luciferases (ALuc) for bioassays, inspired by a sequence alignment of copepod luciferases. Extraction of the consensus amino acids from the alignment enabled us to generate a series of ALucs with unique optical properties and sequential identities that are clearly different from those of any existing copepod luciferase. For example, some ALucs exhibited heat stability, dramatically prolonged optical intensities, broad full width at half maximum, and strong optical intensities. The practical advantages of the luciferases as an optical readout were examined in diverse bioassays, including mammalian two-hybrid assays, live cell imaging, single-chain probes, bioluminescent capsules, and bioluminescent antibodies. We further determine the physical properties of ALucs through bioinformatic analysis and finally discuss detailed issues on the unique properties of ALucs. The present study shows how to create the artificial enzymes with excellent optical properties for bioassays and encourages researchers to fabricate their own unique artificial enzymes with designed properties and functionalities.
[Show abstract][Hide abstract] ABSTRACT: Abiotic and biotic stressors in human cells are often a result of sudden and/or frequent changes in environmental factors. The molecular response to stress involves elaborate modulation of gene expression and is of homeostatic, ecological, and evolutionary importance. Although attention has primarily focused on signaling pathways and protein networks, long non-coding RNAs (ncRNAs) are increasingly involved in the molecular mechanisms associated with responses to cellular stresses. We identified six novel short-lived long ncRNAs (MIR22HG, GABPB-AS1, LINC00152, IDI2-AS1, SNHG15, and FLJ3363) that responded to chemical stressors (cisplatin, cycloheximide, and mercury (II) oxide) in HeLa Tet-off cells. Our results indicate that short-lived long ncRNAs respond to general and specific chemical stressors. The expression levels of the short-lived long ncRNAs were elevated because of prolonged decay rates in response to chemical stressors and interruption of RNA degradation pathways. We propose that these long ncRNAs have the potential to be surrogate indicators of cellular stress responses.
Biochemical and Biophysical Research Communications 09/2013; 439(4). DOI:10.1016/j.bbrc.2013.09.006 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.
[Show abstract][Hide abstract] ABSTRACT: Studies of various mRNAs have revealed that changes in the abundance of transcripts, through mRNA degradation, act as a critical step in the control of various biological pathways. Similarly, the regulation of non-coding RNA (ncRNA) levels is also considered to be important for their biological functions; however, far less is known about the mechanisms and biological importance of ncRNA turnover for the regulation of ncRNA functions. The growth arrest-specific 5 (GAS5) ncRNA accumulates during growth arrest induced by serum starvation and its transcript is degraded by the well characterized nonsense-mediated RNA decay (NMD) pathway. Historically, NMD was discovered as a RNA quality control system to eliminate aberrant transcripts; however, accumulating evidence shows that NMD also regulates the abundance of physiological transcripts. Interestingly, the GAS5 transcript has the ability to bind the glucocorticoid receptor (GR), resulting in the inhibition of its ligand-dependent association with DNA. The GR binds the promoters of various glucocorticoid-responsive genes, including apoptosis-related genes. In this study, we examined whether the RNA degradation pathway can regulate this function of GAS5. We measured the steady-state abundance and the decay rate of GAS5 in UPF1-depleted human cells using the 5'-bromo-uridine immunoprecipitation chase (BRIC) method, an inhibitor-free method for directly measuring RNA stability. We found that levels of the GAS5 transcript were elevated owing to prolonged decay rates in response to UPF1 depletion, and consequently the apoptosis-related genes, cIAP2 and SGK1, were down-regulated. In addition, serum starvation also increased the transcript levels of GAS5 because of prolonged decay rates, and conversely decreased levels of cIAP2 and SGK1 mRNA. Taken together, we found that the RNA degradation pathway can regulate the function of the GAS5 ncRNA in mammalian cells.
PLoS ONE 01/2013; 8(1):e55684. DOI:10.1371/journal.pone.0055684 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A bioluminescent capsule was designed for illuminating cell signaling and protein localization. The capsule consists of four components, namely, a secretion peptide (SP), a luciferase body, a cargo protein (or peptide), and a membrane-localization signal (MLS). Any functional proteins sandwiched between the luciferase body and MLS may be cartable to the plasma membrane (PM), where the capsule waits for outer signals and quickly releases the embedded luciferase in response to the signals. With this strategy of locating the capsule in the PM, the bioluminescence intensity was greatly prolonged and strengthened. A staurosporine (STS)-activated apoptosis signaling was efficiently imaged with the capsule carrying a DEVD peptide. Other functional proteins, such as fluorescent proteins and luciferases, were efficiently transported to the membrane by the capsule. A 60-nm-red-shifted bioluminescence was observed with a capsule fused with other luciferases or fluorescent proteins in living cells. This study gives a new insight regarding how to illuminate cellular signals with bioluminescence in living mammalian cells.
[Show abstract][Hide abstract] ABSTRACT: The taxonomy of the members of the Lactobacillus casei group is complicated because of their phylogenetic similarity and controversial nomenclatural status. In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of ribosomal proteins coded in the S10-spc-alpha operon, termed S10-GERMS, was applied in order to classify 33 sample strains belonging to the L. casei group. A total of 14 types of ribosomal protein genes coded in the operon were first sequenced from four type strains of the L. casei group (L. casei JCM 1134(T), L. paracasei subsp. paracasei JCM 8130(T), L. paracasei subsp. tolerans JCM 1171(T), and L. rhamnosus JCM 1136(T)) together with L. casei JCM 11302, which is the former type strain of 'L. zeae'. The theoretical masses of the 14 types of ribosomal proteins used as biomarkers were classified into five types and compiled into a ribosomal protein database. The observed ribosomal proteins of each strain, identified by MALDI-TOF MS, were categorized into types based on their masses, summarized as ribosomal protein profiles, and they were used to construct a phylogenetic tree. The 33 sample strains, together with seven genome-sequenced strains, could be classified into four major clusters, which coincided precisely with the taxa of the (sub)species within the L. casei group. Three "ancient" strains, identified as L. acidophilus and L. casei, were correctly re-identified as L. paracasei subsp. paracasei by S10-GERMS. S10-GERMS would thus appear to be a powerful tool for phylogenetic characterization, with considerable potential for management of culture collections.
[Show abstract][Hide abstract] ABSTRACT: Perylene, which is composed of five benzene rings, is commonly found in sediments throughout the world at concentrations and distributions that are different from those of other polycyclic aromatic hydrocarbons. The only information available on the origin of perylene comes from 4,9-dihydroxyperylene-3,10-quinone (DHPQ), which originates from fungal component symbiosis or from parasites on plants; however, there is no direct evidence of a mechanism of perylene formation. In this study, we examined the relationship between sedimentary perylene and Cenococcum geophilum (C. geophilum) in a catchment area at Lake Biwa. Sclerotium grains of C. geophilum containing DHPQ were found in this catchment area (approximately 40 balls kg−1 dried soil for >1 mm-ϕ), and small sclerotium grains were frequently found in the sediment. In the sediment sample, we also found broken particles containing perylene, and they had a porous structure characteristic of sclerotium grains. Furthermore, the particles contained DHPQ in different transformation stages to perylene via 3,10-perylenequinone (3,10-PQ). This finding was consistent with results from elemental analysis (oxygen/carbon). Because a remarkable amount of DHPQ originating from C. geophilum also exists in the humic acids of soils and because the inputs of compounds to the lake depend strongly on the rivers, perylene in the Lake Biwa sediment originates mainly from the DHPQ of C. geophilum in its catchment area.
[Show abstract][Hide abstract] ABSTRACT: Copepods are the dominant taxa in zooplankton communities of the ocean worldwide. Although bioluminescence of certain copepods has been known for more than a 100 years, there is very limited information about the structure and evolutionary history of copepod luciferase genes. Here, we report the cDNA sequences of 11 copepod luciferases isolated from the superfamily Augaptiloidea in the order Calanoida. Highly conserved amino acid residues in two similar repeat sequences were confirmed by the multiple alignment of all known copepod luciferases. Copepod luciferases were classified into two groups of Metridinidae and Heterorhabdidae/Lucicutiidae families based on phylogenetic analyses, with confirmation of the interrelationships within the Calanoida using 18S ribosomal DNA sequences. The large diversity in the specific activity of planktonic homogenates and copepod luciferases that we were able to express in mammalian cultured cells illustrates the importance of bioluminescence as a protective function against predators. We also discuss the relationship between the evolution of copepod bioluminescence and the aspects of their ecological characteristics, such as swimming activity and vertical habitat.
[Show abstract][Hide abstract] ABSTRACT: Cortisol is a classical biomarker for the stress levels of human beings. We fabricated highly sensitive bioluminescent probes for salivary cortisol. The following strategies were contrived in the molecular design. Gaussia princeps luciferase (GLuc) was dissected into two fragments, between which an N-terminal-extended ligand binding domain of glucocorticoid receptor (GR HLBD), named Simgr4, was inserted. First, this unique single-chain probe was then situated downstream of a glucocorticoid response element (GRE) promoter in a reporter-gene system for constructing two ON-OFF switches for cortisol. Second, a circularly permutated (CP) variant of Simgr4 was formulated. The reporter-gene system exerted an improved signal-to-background (S/B) ratio of 8.5 to cortisol. Furthermore, a circularly permutated (CP) variant of Simgr4 exerted a 10× enhanced detection limit to cortisol and a long dynamic range from 10(-9) to 10(-6) M cortisol, covering all of the normal clinical ranges of serum, urine, and saliva. This optimized probe successfully determined daily fluctuations of salivary cortisol and the correlations with those by ELISA. This study is the first to investigate the contribution of the HLBD of a nuclear receptor and multiple ON-OFF switches for molecular probes and salivary cortisols.
[Show abstract][Hide abstract] ABSTRACT: Rhodococcus erythropolis strains characterized as antibiotic producers can be classified into three groups according to their antibiotic spectrum and growth compatibility. Due to their high genotypic similarity, the taxonomic relationship of these strains has not been elucidated. In this study, ribosomal protein profiling using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) was employed to classify twenty-one strains of R. erythropolis (15 antibiotic producers and 6 non-antibiotic producers). In the first step in this method, a total of 30 intense peaks observed for purified ribosomal subunit proteins of the type strain (R. erythropolis JCM 3201(T)) were selected as the reference peaks. The mass spectra observed for the cell lysates of each sample strain were then checked as to whether peaks were observed at the same masses of the reference peaks. The results of peak matching were processed by cluster analysis, generating a dendrogram. Four major clusters of the R. erythropolis strains corresponded to three antibiotic groups and the non-antibiotic group. Furthermore, the topology of the dendrogram was highly comparable with the phylogenetic tree based on DNA gyrase subunit B gene (gyrB) sequencing. These results indicate that our proposed ribosomal protein profiling method using MALDI-MS is a potentially reliable and sufficiently high-throughput technique for the taxonomic analysis of closely related bacterial strains without using DNA sequence information.
Journal of Bioscience and Bioengineering 11/2009; 108(4):348-53. DOI:10.1016/j.jbiosc.2009.04.010 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the current study, we investigated photoinduced electron transfer from chromatophore vesicles or intact cells of Rhodobacter sphaeroides to an exogenous artificial electron acceptor (2,5-dichloro-1,4-benzoquinone, DCBQ) by amperometric measurement using a rotating-disk electrode. We found that electrons transfer from chromatophores or intact cells to the working electrode via DCBQ in the presence of light. Application of a positive potential revealed that chromatophores or intact cells adsorbed to the surface of the working electrode. Photoreduction of DCBQ occurred by Michaelis–Menten type kinetics with respect to the photon flux intensity and DCBQ concentration, allowing Michaelis constants and maximum photoinduced electron transfer reaction rates to be calculated. Finally, we show that the system using chromatophores, which are more stable than intact cells, can be used to detect herbicides.
[Show abstract][Hide abstract] ABSTRACT: We have developed a novel method for microbial community analysis of bacterial 16S rRNAs based on affinity capillary electrophoresis using 16S rRNA-conjugated magnetic beads. We called this method magnetic beads affinity capillary electrophoresis (MB-ACE) which can be used for sequential and quantitative analysis of 16S rRNA. In this method, RNA extracted from a microbial community is biotin-modified and mixed with streptavidin-modified paramagnetic beads. This mixture is then injected into a capillary and localized in the middle of the capillary using a magnet held adjacent to the capillary. Subsequently, a fluorescent-labeled probe to detect the target 16S rRNA is injected into the capillary, and voltage is applied. The probe trapped on the RNA is dissociated by formamide and detected at its anodic end by measuring the fluorescence. Next, another fluorescent probe is injected, and thus the target 16S rRNA in the sample is quantified one by one. MB-ACE was used for the quantification of the 16S rRNAs of Escherichia coli and Pseudomonas putida in samples that were prepared by mixing RNA extracted from activated sludge and 16S rRNAs prepared by in vitro transcription. The two types of 16S rRNAs were quantified, indicating that MB-ACE can be used for sequential quantitative analysis of bacterial 16S rRNAs.
Journal of Bioscience and Bioengineering 07/2009; 107(6):662-7. DOI:10.1016/j.jbiosc.2009.02.004 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have developed an 8-ch capillary-based dispensing workstation with a variable capillary pitch mechanism. The capillary intervals can be varied from 1 to 9 mm to dispense different solutions simultaneously at an arbitrary dispensing pitch, allowing direct dispensing from microplates to integrated analytical systems. To evaluate the precision of its dispensing performance, droplets of Rhodamine G dye were dispensed onto glass slides and the values of the optical volume were analyzed. The error in the dispensed volume proved to be 0.54 nL when dispensing 20 nL. In dispensing small volumes, the volume error for this workstation was found to be about 100-fold less than that seen in conventional dispensers. Even highly viscous solutions containing 50% glycerol could be dispensed with precision. Rapid dispensing was also achieved. Moreover, the application of the workstation to preparing addressable 8 x 12 microsensor array chips was demonstrated, providing an independent and reproducible spot array.
[Show abstract][Hide abstract] ABSTRACT: Psychrotrophic lactic acid bacteria strains potentially used for the fermentation of meat were characterized by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using ribosomal proteins as biomarkers. The sample strains of a commercial starter culture (strain D-1001) and 15 similar strains were thought to be Lactobacillus sakei, based on their biochemical properties and the 16S rRNA partial gene sequence. By comparing the mass spectra of cell 1,sates to the calculated masses of ribosomal proteins of genome-sequenced Lactobacillus sakei 23K, all sample strains were definitely identified as L. sakei. Sample strains were further classified into 4 groups based on the mass differences of the 16 biomarkers selected from ribosomal subunit proteins, which could not be discriminated using the 16S rRNA partial gene sequence analysis.
[Show abstract][Hide abstract] ABSTRACT: A new method for phylogenetic classification of bacterial strains 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 approximately 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 MALDI-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 strain. 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 sub-unit 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Proteome Research 11/2007; 6(10):3899-907. DOI:10.1021/pr070218l · 4.25 Impact Factor