[Show abstract][Hide abstract]ABSTRACT: Rapid, simple, and low-cost screening procedures are necessary for the detection of harmful compounds in the effluent that flows out of point sources such as industrial outfall. The present study investigated the effects on a novel sensor of harmful compounds such as KCN, phenol, and herbicides such as 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine (atrazine), and 2-N-tert-butyl-4-N-ethyl-6-methylsulfanyl-1,3,5-triazine-2,4-diamine (terbutryn). The sensor employed an electrode system that incorporated the photocurrent of intra-cytoplasmic membranes (so-called chromatophores) prepared from photosynthetic bacteria and linked using carbon paste electrodes. The amperometric curve (photocurrent-time curve) of photo-induced electron transfer from chromatophores of the purple photosynthetic bacterium Rhodobacter sphaeroides to the electrode via an exogenous electron acceptor was composed of two characteristic phases: an abrupt increase in current immediately after illumination (I₀), and constant current over time (Ic). Compared with other redox compounds, 2,5-dichloro-1,4-benzoquinone (DCBQ) was the most useful exogenous electron acceptor in this system. Photo-reduction of DCBQ exhibited Michaelis-Menten-like kinetics, and reduction rates were dependent on the amount of DCBQ and the photon flux intensity. The Ic decreased in the presence of KCN at concentrations over 0.05 μM (=μmol·dm(-3)). The I₀ decreased following the addition of phenol at concentrations over 20 μM. The Ic was affected by terbutryn at concentrations over 10 μM. In contrast, DCMU and atrazine had no effect on either I₀ or Ic. The utility of this electrode system for the detection of harmful compounds is discussed.
[Show abstract][Hide abstract]ABSTRACT: Because of the limitations of whole animal testing approaches for toxicological assessment, new cell-based assay systems have been widely studied. In this study, we focused on two biological products for toxicological assessment: mouse embryonic stem cells (mESCs) and long noncoding RNAs (lncRNAs). mESCs possess the abilities of self-renewal and differentiation into multiple cell types. LlncRNAs are an important class of pervasive non-protein-coding transcripts involved in the molecular mechanisms associated with responses to chemicals. We exposed mESCs to p-dichlorobenzene (p-DCB) for 1 or 28 days (daily dose), extracted total RNA, and performed deep sequencing analyses. The genome-wide gene expression analysis indicated that mechanisms modulating proteins occurred following acute and chronic exposures, and mechanisms modulating genomic DNA occurred following chronic exposure. Moreover, our results indicate that three novel lncRNAs (Snora41, Gm19947, and Scarna3a) in mESCs respond to p-DCB exposure. We propose that these lncRNAs have the potential to be surrogate indicators of p-DCB responses in mESCs.
No preview · Article · Mar 2016 · Journal of Bioscience and Bioengineering
[Show abstract][Hide abstract]ABSTRACT: The effect of the sp(2)/sp(3) ratio in an unbalanced magnetron sputtered nanocarbon film electrode was studied for determining Cd(2+) and Pb(2+) by anodic stripping voltammetry (ASV). The signal-to-noise ratio in the ASV measurement improved as the sp(3) concentration in the carbon film increased because the noise current decreased with the increasing sp(3) concentration. The detection limits with a carbon film containing 50% sp(3) were 0.25 and 1.0 μg L(-1) for Cd(2+) and Pb(2+) with high repeatability (Cd: 4.6% and Pb: 6.4%, n = 3). For a real sample measurement, a pretreatment system combining a photooxidation reactor and a cation exchange column was used to eliminate the interference from EDTA and Cu(2+), which forms a stable complex or alloy with Cd(2+) and Pb(2+). More than 99% of the interference was eliminated, and accurate signal currents for Cd(2+) and Pb(2+) were successfully obtained with the pretreatment system.
[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.
No preview · Article · Nov 2014 · Journal of Bioscience and Bioengineering
[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.
[Show abstract][Hide abstract]ABSTRACT: In a dispensing apparatus for dispensing a liquid, such as a sample solution, on a substrate, such as a glass slide, the dispensing apparatus includes a capillary provided with a distal end and a proximal end, a pump unit configured to pump an operating liquid into the capillary and to pump the operating liquid out of the capillary, and a controller configured to control the pump unit so as to change a position of a liquid surface of the operating liquid in the capillary so that a predetermined volume of liquid is suctioned from the distal end into the capillary and the liquid suctioned in the capillary is discharged from the distal end. As a result, it is possible to precisely dispense extremely small volume amounts of liquids such as a nanoliter.
[Show abstract][Hide abstract]ABSTRACT: A spotter that includes a plurality of spotting heads, each of the plurality of spotting heads having a discharging portion at a tip portion, the plurality of spotting heads form an m×n array (m, n>1) with m spotting heads arranged lengthwise and n spotting heads arranged crosswise; and a pitch varying mechanism configured to vary an array pitch of the plurality of spotting heads arrayed in a lengthwise direction and an array pitch of the plurality of spotting heads arrayed in a crosswise direction.
[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.
No preview · Article · Jan 2014 · Solvent Extraction Research and Development Japan
[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.
No preview · Article · Nov 2013 · Bioconjugate Chemistry
[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.
No preview · Article · Sep 2013 · Biochemical and Biophysical Research Communications
[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.
[Show abstract][Hide abstract]ABSTRACT: Knockdown of UPF1 increases the expression level of GAS5, and decreases the glucocorticoid-responsive genes. HEK293 cells were treated with a control siRNA or with a siRNA targeting UPF1. The expression levels of indicated genes in control cells (black bar) and in UPF1-depleted cells using UPF1 siRNA (2) (grey bar) was determined by RT-qPCR. The levels of GAPDH and ACTB were used for normalization. Values represent mean±SD obtained from three independent experiments (**P<0.01, student's test).
[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.
No preview · Article · Oct 2012 · Bioconjugate Chemistry
[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.
No preview · Article · Oct 2012 · Systematic and Applied Microbiology
[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.
Full-text · Article · Oct 2012 · Geochimica et Cosmochimica Acta
[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.
Full-text · Article · Mar 2012 · Molecular Biology and Evolution
[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.
No preview · Article · Aug 2011 · Bioconjugate Chemistry
[Show abstract][Hide abstract]ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.