• Journal of Microbiology and Biotechnology
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• v.21 no.12
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• pp.1336-1344
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• 2011

This study details and examines a novel ligation-mediated polymerase chain reaction (LM-PCR) method. Named the LM-PCR/Shifter, it relies on the use of a Class IIS restriction enzyme giving restriction fragments with different 4-base, 5' overhangs, this being the Shifter, and the ligation of appropriate oligonucleotide adapters. A sequence of 4-base, 5' overhangs of the adapter and a 4-base sequence of the 3' end of the primer(s) determine a subset of the genomic restriction fragments, which are amplified by PCR. The method permits the differentiation of bacterial species strains on the basis of the different DNA band patterns obtained after electrophoresis in polyacrylamide gels stained with ethidium bromide and visualized in UV light. The usefulness of the LM-PCR/Shifter method for genotyping is analyzed by a comparison with the restriction endonuclease analysis of chromosomal DNA by the pulsed-field gel electrophoresis (REA-PFGE) and PCR melting profile (PCR MP) methods for isolates of clinical origin. The clustering of the LM-PCR/Shifter fingerprinting data matched those of the REA-PFGE and PCR MP methods. We found that the LM-PCR/Shifter is rapid, and offers good discriminatory power and excellent reproducibility, making it a method that may be effectively applied in epidemiological studies.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.1007-1014
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• 2015

Plectasin, the first defensin extracted from a fungus (the saprophytic ascomycete Pseudoplectania nigrella), is attractive as a prospective antimicrobial agent. The purpose of this study was to establish a bacterium-based production system and evaluate the antimicrobial activity of the resulting plectasin. A gene encoding plectasin, with the codon preference of Escherichia coli, was optimized based on its amino acid sequence, synthesized using genesplicing with overlap extension PCR, and inserted into the expression vector pGEX-4T-1. The fusion protein was expressed in the soluble fraction of E. coli and purified using glutathione Stransferase affinity chromatography. Plectasin was cleaved from the fusion protein with thrombin and purified by ultrafiltration. The purified plectasin showed strong, concentrationdependent antimicrobial activity against gram-positive bacteria, including antibiotic-resistant bacteria, especially penicillin-resistant Enterococcus faecium. This antimicrobial activity was equal to chemically synthesized plectasin and was maintained over a wide range of pH and temperatures. This soluble recombinant expression system in E. coli is effective for producing plectasin at a relatively lower cost, and higher purity and efficiency than prior systems, and might provide a foundation for developing a large-scale production system. Overall, plectasin shows potential as a novel, high-performance, and safe antibiotic for the treatment of refractory diseases caused by drug-resistant bacterial strains.

• Journal of Microbiology and Biotechnology
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• v.17 no.11
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• pp.1875-1879
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• 2007

Strain $CH7^T$, a pale yellow-pigmented bacterium and new isolate from deep subsurface water of the South Coast of Korea, was subjected to a polyphasic taxonomic study. $CH7^T$ grew between 5 and $37^{\circ}C$, pH 5.3-10.5, and tolerated up to 13% NaCl. A phylogenetic analysis based on 16S rRNA gene sequences showed that strain $CH7^T$ was associated with the genus Arthrobacter and phylogenetically closely related to the type strains Arthrobacter tumbae (99.4%) and Arthrobacter parietis (99.1%). However, DNA-DNA hybridization experiments revealed 2.1% and 12% between strain $CH7^T$ and Arthrobacter tumbae and Arthrobacter parietis, respectively. Thus, the phenotypic and phylogenetic differences suggested that $CH7^T$ should be placed in the genus Arthrobacter as a novel species, for which the name Arthrobacter subterraneus sp. novo is proposed. In addition, the type strain for the new species is $CH7^T$ ($=KCTC\;9997^T=DSM\;17585^T$).

• Journal of Microbiology and Biotechnology
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• v.23 no.10
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• pp.1381-1385
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• 2013

The centipede Scolopendra subpinipes mutilans is a medicinally important arthropod species. However, its transcriptome is not currently available and transcriptome analysis would be useful in providing insight into a molecular level approach. Hence, we performed de novo RNA sequencing of S. subpinipes mutilans using next-generation sequencing. We generated a novel peptide (scolopendrasin II) based on a SVM algorithm, and biochemically evaluated the in vitro antimicrobial activity of scolopendrasin II against various microbes. Scolopendrasin II showed antibacterial activities against gram-positive and -negative bacterial strains, including the yeast Candida albicans and antibiotic-resistant gram-negative bacteria, as determined by a radial diffusion assay and colony count assay without hemolytic activity. In addition, we confirmed that scolopendrasin II bound to the surface of bacteria through a specific interaction with lipoteichoic acid and a lipopolysaccharide, which was one of the bacterial cell-wall components. In conclusion, our results suggest that scolopendrasin II may be useful for developing peptide antibiotics.

• Journal of Microbiology and Biotechnology
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• v.23 no.11
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• pp.1536-1543
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• 2013

Proteases produced by Xenorhabdus are known to play a significant role in virulence leading to insect mortality. The present study was undertaken to purify and characterize protease from Xenorhabdus indica, an endosymbiont of nematode Steinernema thermophilum, and to decipher its role in insect mortality and its efficacy to control Helicoverpa armigera. A set of 10 strains of Xenorhabdus isolated from different regions of India were screened for protease activity on the basis of zone of clearing on gelatin agar plates. One potent strain of Xenorhabdus indica was selected for the production of protease, and the highest production (1,552 U/ml) was observed at 15-18 h of incubation at $28^{\circ}C$ in soya casein digest broth. The extracellular protease was purified from culture supernatant using ammonium sulfate precipitation and ion-exchange chromatography. The enzyme was further characterized by SDS-PAGE and zymography, which confirmed the purity of the protein and its molecular mass was found to be ~52 kDa. Further MALDI-TOF/TOF analysis and effect of metal chelating agent 1,10-phenanthrolin study revealed the nature of the purified protease as a secreted alkaline metalloprotease. The bioefficacy of the purified protease was also tested against cotton bollworm (Helicoverpa armigera) and resulted in $67.9{\pm}0.64%$ mortality within one week. This purified protease has the potential to be developed as a natural insecticidal agent against a broad range of agriculturally important insects.

• KSBB Journal
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• v.29 no.3
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• pp.131-138
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• 2014

In this study, we isolated two novel chitinase producing bacterial strains from yellow loess samples collected from Jullanamdo province. The chitinase producing bacteria were isolated based on the zone size of clearance in the chitin agar plates. Both of them were gram positive, rod ($2{\sim}3{\times}0.3{\sim}0.4{\mu}m$), spore-forming, and motility positive. They were facultative anaerobic, catalase positive and hydrolyzed starch, gelatin, and casein. From the 16s rRNA gene sequence analysis, the isolates were labeled as Bacillus licheniformis KYLS-CU01 and B. licheniformis KYLS-CU02. The isolates showed higher extracellular chitinase activities than B. licheniformis ATCC 14580 as a control. The optimum temperature and pH for chitinase production were $40^{\circ}C$ and pH 7.0, respectively. Response Surface Methodology (RSM) was used to optimize the culture medium for efficient production of the chitinase. Under this optimal condition, 1.5 times higher chitinase activity of B. licheniformis KYLS-CU02 was obtained. Extracellular chitinases of the two isolates were purified through ammonium sulfate precipitation and anion-exchange DEAE-cellulose column chromatography. The specific activities of purified chitinase from B. licheniformis KYLS-CU01 and B. licheniformis KYLS-CU02 were 7.65 and 5.21 U/mg protein, respectively. The molecular weights of the two purified chitinases were 59 kDa. Further, the purified chitinase of B. licheniformis KYLS-CU01 showed high antifungal activity against Fusarium sp.. In conclusion, these two bacterial isolates can be used as a biopesticide to control pathogenic fungi.

• Ceramist
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• v.23 no.1
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• pp.89-100
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• 2020

Abnormally large electromechanical strain properties have been reported in bismuth-based piezoelectric ceramics, which cast a promise for replacing the market-dominating PZT-based piezoelectric ceramics in actuator applications. In spite of these large strains in bismuth-based piezoelectric ceramics, there still remains a critical issue for its safe transfer to practical applications, representatively, a relatively high operating field required to obtain the large strain properties. To overcome the challenge, much attention has been paid to so-called 0-3(or 3-0) type ceramic/ceramic composite approach to better tailoring the strain properties of bismuth-based piezoelectric ceramics. The approach turns out to be highly effective, leading to a drastic decrease in the operating electric field for these materials. Besides, both extensive and intensive search for the related mechanism revealed that the reduction in the operating electric field is largely due to the contribution from polarization coupling or strain coupling model between two different ceramics. This article reviews the status of the art in the development of novel ceramic/ceramic composites to make large incipient piezostrains in bismuth-based lead-free piezoelectric ceramics practical.

• Food Science and Biotechnology
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• v.15 no.6
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• pp.851-855
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• 2006

Acarviosine-glucose (AcvGlc) is an ${\alpha}$-glucosidase inhibitor and has similar inhibitory activity to acarbose in vitro. We synthesized AcvGlc by treating acarbose with Bacillus stearothermophilus maltogenic amylase and fed C57BL/6J and db/db mice with diets containing purified AcvGlc and acarbose for 1 week. AcvGlc (50 and 100 mg/100 g diet) significantly reduced plasma glucose and triglyceride levels in db/db mice by 42 and 51 %, respectively (p<0.0001). The hypoglycemic and hypotriglyceridemic effects of AcvGlc were slightly, but significantly, greater than those seen with acarbose treatment (p<0.0001) in C57BL/6J mice. In an oral glucose tolerance test, glucose tolerance was significantly improved at all time points (p<0.01). The expression of two novel glucose transporters (GLUTs), GLUT10 and GLUT12, were examined by Western blot analysis. GLUT10 was markedly increased in the db/db livers. After AcvGlc treatment, the expression of hepatic GLUT10 was decreased whereas intestinal GLUT12 was significantly increased in both strains of mice. Our results show that AcvGlc improves plasma lipid and glucose metabolism slightly more than acarbose. Regulation of hepatic GLUT10 and intestinal GLUT12 may be important in controlling blood glucose levels.

• Toxicological Research
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• v.24 no.2
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• pp.151-159
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• 2008

Rhus verniciflua Stokes(RVS), one of traditional medicinal plants in Asia, was found to have pharmacological activities such as antioxidative and antiapoptotic effects, raising the possibility for the development of a novel class of anti-cancer drugs. Thus, potential genotoxic effects of RVS in three short-term mutagenicity assays were investigated, which included the Ames assay, in vitro Chromosomal aberration test, and the in vivo Micronucleus assay. In Ames test, the addition of RVS water extracts at doses from 313 up to 5000 mg/plate induced an increase more than 2-fold over vehicle control in the number of revertant colonies in TA98 and TA1537 strains for detecting the frame-shift mutagens. The similar increase in reversion frequency was observed after the addition of RVS ethanol extracts. To assess clastogenic effect, in vitro chromosomal aberration test and in vivo micronucleus assay were performed using Chinese hamster lung cells and male ICR mice, respectively. Both water and ethanol extracts from RVS induced significant increases in the number of metaphases with structural aberrations mostly at concentrations showing the cell survival less than 60% as assessed by in vitro CA test. Also, there was a weak but statistically significant increase in number of micronucleated polychromatic erythrocytes(MNPCEs) in mice treated with water extract at 2000 mg/kg while ethanol extracts of RVS at doses of up to 2000 mg/kg did not induce any statistically significant changes in the incidence of MNPCEs. Therefore, our results lead to conclusion that RVS acts as a genotoxic material based on the available in vitro and in vivo results.

• Structural Engineering and Mechanics
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• v.75 no.2
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• pp.193-209
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• 2020

This paper presents a new hyperbolic shear deformation plate theory including the stretching effect for free vibration of the simply supported functionally graded plates in thermal environments. The theory accounts for parabolic distribution of the transverse shear strains and satisfies the zero traction boundary conditions on the surfaces of the plate without using shear correction factors. This theory has only five unknowns, which is even less than the other shear and normal deformation theories. The present one has a new displacement field which introduces undetermined integral variables. Material properties are assumed to be temperature-dependent, and graded in the thickness direction according to a simple power law distribution in terms of the volume power laws of the constituents. The equation of motion of the vibrated plate obtained via the classical Hamilton's principle and solved using Navier's steps. The accuracy of the proposed solution is checked by comparing the present results with those available in existing literature. The effects of the temperature field, volume fraction index of functionally graded material, side-to-thickness ratio on free vibration responses of the functionally graded plates are investigated. It can be concluded that the present theory is not only accurate but also simple in predicting the natural frequencies of functionally graded plates with stretching effect in thermal environments.