A short peptide designed from late-embryogenesis abundant protein enhances acid tolerance in Escherichia coli

Unsuitable pH is a major limiting factor for all organisms, and a low pH can lead to organism death. Late embryogenesis abundant (LEA) peptides confer tolerance to abiotic stresses including salinity, drought, high and low temperature, and ultraviolet radiation same as the LEA proteins from which they originate. In this study, LEA peptides derived from group 3 LEA proteins of Polypedilum vanderplanki were used to enhance low pH tolerance. Recombinant Escherichia coli BL21 (DE3) cells expressing the five designed LEA peptides were grown at pH 4, 3, and 2. The transformants showed higher growth capacity at low pH as compared to control cells. These results indicate that LEA peptide could prevent E. coli cell death under low pH conditions

In vivo expression of a short peptide designed from late embryogenesis abundant protein for enhancing abiotic stress tolerance in Escherichia coli

In vivo functional analyses of a late embryogenesis abundant (LEA) short peptide expressed in recombinant Escherichia coli BL21 (DE3) were carried out under abiotic stress (salt, heat, and cold) conditions. Our LEA peptide was derived from the Polypedilum vanderplanki group 3 LEA protein based on distinctive conserved amino acid motif sequences. We focused on high-salt (5% and 7% NaCl) concentrations to evaluate the functional relevance of the peptide under abiotic salt stress. E. coli transformants expressing the LEA peptide showed higher cell viability than the control not expressing the peptide when transferred to a medium containing 5% and 7% NaCl; cells expressing LEA peptide showed a higher number of colony-forming units per dilution under the high salt stress condition. Moreover, expression of the LEA peptide resulted in greater cell survival under heat (48 °C) and cold (4 °C) stress. These results suggest that LEA short peptide co-expression could be useful for developing genetically modified organisms and in applications to prevent E. coli cell death under high salt, heat, and cold stress

Development of New Heat Treatment Method to Impart High Creep Strength and High Toughness to Rotor Material for Condensing Steam Turbine

LectureHigher temperatures in the HP section of the condensing Turbine requires rotor with high creep strength while low temperatures in the LP section require the rotor to have high toughness. The design basis for current rotor material, Ni-1.25Cr-Mo-V forged steel was high toughness at low temperatures. This led to rotor having insufficient creep strength in very high temperature region around control stage which limited the maximum allowable temperature of inlet steam thereby limiting the efficiency of the Turbine. This paper highlights the development of new heat treatment method to improve Ni-2.25Cr-Mo-V forged steel (10325MTE) as rotor material. To improve the mechanical properties of the material, heat treatment simulations were performed. A series of tests were done on the rotor. These tests evaluated the creep strength at high temperatures and toughness low temperatures. SCC (Stress Corrosion Cracking) susceptibility was evaluated in an accumulated corrosive environment using SSRT (Slow Strain Rate Test)

Artificial-enzyme gel membrane-based biosurveillance sensor with high reproducibility and long-term storage stability

We propose that the most sophisticated strategy for primary biosurveillance is to exploit structural commonality through the detection of biologically relevant phosphoric substances. A novel assay, an artificial-enzyme membrane was designed and synthesized for sensor fabrication. This artificial-enzyme catalyzes the hydrolysis of the diphosphoric acid anhydride structure. This structure-selective, albeit not molecule-selective, catalytic hydrolysis was successfully coupled with amperometric detection. Since the catalytic reaction produces a dephosphorylation product (PO43−), it can be reduced by an electrode potential of −250 mV vs. Ag/AgCl. Owing to the structural selectivity of the artificial-enzyme membrane, the sensor can detect biological phosphoric substances comprehensively that have the diphosphoric acid anhydride structure. The sensor successfully determined various biological phosphoric substances at concentrations in the micromolar (µM) to millimolar (mM) range, and it showed good functional stability and reproducibility in terms of sensor responses. This sensor was used to detect Escherichia coli lysed by heat treatment, and the response increased with increasing bacterial numbers. This unique technique for analyzing molecular commonality can be applied to the surveillance of biocontaminants, e.g. microorganisms, spores and viruses. Artificial-enzyme-based detection is a novel strategy for practical biosurveillance in the front line

ベンゼン系化合物の生物学的簡易測定システムの構築に関する研究

取得学位：博士(工学)，学位授与番号：博甲第574号，学位授与年月日：平成15年3月25日,学位授与年：200

Escherichia coli tolerance of ultraviolet radiation by in vivo expression of a short peptide designed from late embryogenesis abundant protein

Ultraviolet (UV) radiation causes damage in all living organisms, including DNA damage that leads to cell death. Herein, we provide a new technique for UV radiation protection through intracellular short peptide expression. The late embryogenesis abundant (LEA) peptide, which functions as a shield that protects macromolecules from various abiotic stress, was obtained from the Polypedilum vanderplanki group 3 LEA protein. Recombinant Escherichia coli BL21 (DE3) expressing functional LEA short peptide in vivo were exposed to UVA and UVC radiation for 4, 6, and 8 h. E. coli transformants expressing the LEA peptide showed higher cell viability under both UVA and UVC treatment at all time points as compared with that of the control. Furthermore, the cells expressing LEA peptide showed a higher number of colony-forming units per dilution under UVA and UVC treatment. These results suggested that expression of the short peptide could be useful for the development of genetically modified organisms and in applications that require resilience of organisms to UV radiation

Development of New Heat Treatment Method to Impart High Creep Strength and High Toughness to Rotor Material for Condensing Steam Turbine

LectureHigher temperatures in the HP section of the condensing Turbine requires rotor with high creep strength while low temperatures in the LP section require the rotor to have high toughness. The design basis for current rotor material, Ni-1.25Cr-Mo-V forged steel was high toughness at low temperatures. This led to rotor having insufficient creep strength in very high temperature region around control stage which limited the maximum allowable temperature of inlet steam thereby limiting the efficiency of the Turbine. This paper highlights the development of new heat treatment method to improve Ni-2.25Cr-Mo-V forged steel (10325MTE) as rotor material. To improve the mechanical properties of the material, heat treatment simulations were performed. A series of tests were done on the rotor. These tests evaluated the creep strength at high temperatures and toughness low temperatures. SCC (Stress Corrosion Cracking) susceptibility was evaluated in an accumulated corrosive environment using SSRT (Slow Strain Rate Test)

IDENTIFICATION OF MYCOSPORINE-LIKE AMINO ACIDS AND EXPRESSION OF 3-DEHYDROQUINATE SYNTHASE GENE IN UV RADIATIONS-INDUCED Deinococcus radiodurans R1

Mycosporine-like amino acids (MAAs) are a group of more than 40 metabolites originated from 4-deoxygadusol featuring antioxidants, growth stimulation, and UV protective properties in many microorganisms. In D. radiodurans R1, 3-dehydroquinate synthase (DHQS) gene annotated in chromosome 1 encodes the precursor for all MAAs. In this study, a significant amount of MAAs were identified in D. radiodurans R1 after treatment with a different type of UV radiations, namely; the low energy UVA (360 nm) 6W and 100 W, and high energy UVC (254 nm) 6W at a period of 12 to 48 hours. The total RNA and MAAs were isolated from the UV-treated D. radiodurans R1. RT-qPCR experiment of the DHQS gene resulted in a significant increase of expression. Consequently, specific MAAs were identified using time-of-flight mass spectrometry (TOF-MS). They are mycosporine-taurine, mycosporine-glutamine, mycosporine-glutaminol, mycosporine-glutaminol-glucoside, mycosoprine-glycine, mycosporine-2-glycine, mycosporine-glycine:glutamic acid, shinorine, mycosporine-methylamine:serine, palythine-serine, and palythinol. The results suggested that these compounds play essential roles in D. radiodurans R1 radio-tolerance especially mycosporine-methylamine:serine and palythine-serine. This study can help to further understand the mechanism of radiation resistance in D. radiodurans R1, and its potential to be utilized as protective compound against radiation risk

Gold Nanoparticles Functionalized with Peptides for Specific Affinity Aggregation Assays of Estrogen Receptors and Their Agonists

Nuclear receptors regulate the transcription of genes and various functions such as development, differentiation, homeostasis, and behavior by formation of complexes with ligand and co-activator. Recent findings have shown that agonists of a ligand may have a toxic effect on cellular/tissular function through improper activation of nuclear receptors. In this study, a simple assay system of hetero-complexes of three different molecules (estrogen receptor, ligand, and co-activator peptide) has been developed. This assay system employs functionalized gold nanoparticles (GNPs: 15 nm in diameter). The surfaces of the GNPs were modified by a 12- or 20-amino-acid peptide that contains the sequence of co-activator for activating nuclear receptor by an agonist ligand. Owing to the affinity of the peptide, the functionalized GNPs aggregate faster when the nuclear receptor and the agonist ligand are also present. The aggregation of GNPs can be identified by shifts in adsorption spectrum, which give information about the specificity of agonist ligands. Similarly, this spectrum shift can measure concentration of known agonist ligand. This simple agonist screening will be employed as high through-put analysis (HTA) in the discovery of drugs that act through nuclear receptors

Interaction study of peptide-PAMAM as potential bio-nanogate for detecting anti-hepatitis B surface antigen

Bio-nanogate involves synthesized or natural molecules as a 'gate' towards bioreceptors and responds upon the presence of targeted analytes in nanoscale dimension. Development of bio-nanogate improves analyte selectivity and signal response across various types of biosensors. The versatility of PAMAM dendrimers to form conjugates with guest molecules, such as proteins can be utilized in forming a bio-nanogate. PAMAM interaction with peptide bioreceptor for antibody detection is of interest in this study. This study investigated the interaction of synthesized immunogenic 'a' determinant (aD) region of hepatitis B virus surface antigen (HBsAg) with PAMAM G4 and anti-HBsAg antibody, as a potential bio-nanogate for anti-HBsAg detection. The aD peptide fused with maltose binding protein (MBP), was confirmed with Western blotting. Nano-Differential Scanning Fluorimetry (nano-DSF) study revealed that the interaction of MBP-aD with anti-HBsAg indicated a higher thermal stability as compared to its interaction with PAMAM G4. Electrochemical impedance spectroscopy showed that a higher binding constant of MBP-aD interaction with anti-HBsAg (0.92 μM-1) was observed at maximum saturation, as compared with PAMAM G4 (0.07 μM-1). Thermodynamic parameters demonstrated that MBP-aD interacted with anti-HBsAg and PAMAM G4, through van der Waals and hydrogen bonding. These analyses suggest that the weak interaction of MBP-aD and PAMAM G4 may form a potential bio-nanogate. It is hypothesized that the presence of anti-HBsAg has a higher affinity towards MBP-aD which may displace PAMAM G4 in the anti-HBsAg detection system. This interaction study is crucial as an initial platform of using peptide-PAMAM as a bio-nanogate in an antibody detection system