• Journal of Pharmacopuncture
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• v.19 no.3
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• pp.213-224
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• 2016

Objectives: Ginseng Rh2+ is enzyme-treated ginseng extract containing high amounts of converted ginsenosides, such as compound k, Rh2, Rg3, which have potent anticancer activity. We conducted general and genetic toxicity tests to evaluate the safety of ginseng Rh2+. Methods: An acute oral toxicity test was performed at a high-level dose of 4,000 mg/kg/day in Sprague-Dawley (SD) rats. A 14-day range-finding study was also conducted to set dose levels for the 90-day study. A subchronic 90-day toxicity study was performed at dose levels of 1,000 and 2,000 mg/kg/day to investigate the no-observed-adverse-effect level (NOAEL) of ginseng Rh2+ and target organs. To identify the mutagenic potential of ginseng Rh2+, we conducted a bacterial reverse mutation test (Ames test) using amino-acid-requiring strains of Salmonella typhimurium and Escherichia coli (E. coli), a chromosome aberration test with Chinese hamster lung (CHL) cells, and an in vivo micronucleus test using ICR mice bone marrow as recommended by the Korean Ministry of Food and Drug Safety. Results: According to the results of the acute oral toxicity study, the approximate lethal dose (ALD) of ginseng Rh2+ was estimated to be higher than 4,000 mg/kg. For the 90-day study, no toxicological effect of ginseng Rh2+ was observed in body-weight changes, food consumption, clinical signs, organ weights, histopathology, ophthalmology, and clinical pathology. The NOAEL of ginseng Rh2+ was established to be 2,000 mg/kg/day, and no target organ was found in this test. In addition, no evidence of mutagenicity was found either on the in vitro genotoxicity tests, including the Ames test and the chromosome aberration test, or on the in vivo in mice bone marrow micronucleus test. Conclusion: On the basis of our findings, ginseng Rh2+ is a non-toxic material with no genotoxicity. We expect that ginseng Rh2+ may be used as a novel adjuvant anticancer agent that is safe for long-term administration.

• Journal of Microbiology and Biotechnology
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• v.22 no.2
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• pp.184-189
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• 2012

We sought to breed an industrially useful yeast strain, specifically an ethanol-tolerant yeast strain that would be optimal for ethanol production, using a novel breeding method, called genome reconstruction, based on chromosome splitting technology. To induce genome reconstruction, Saccharomyces cerevisiae strain SH6310, which contains 31 chromosomes including 12 artificial mini-chromosomes, was continuously cultivated in YPD medium containing 6% to 10% ethanol for 33 days. The 12 mini-chromosomes can be randomly or specifically lost because they do not contain any genes that are essential under high-level ethanol conditions. The strains selected by inducing genome reconstruction grew about ten times more than SH6310 in 8% ethanol. To determine the effect of mini-chromosome loss on the ethanol tolerance phenotype, PCR and Southern hybridization were performed to detect the remaining mini-chromosomes. These analyses revealed the loss of mini-chromosomes no. 11 and no. 12. Mini-chromosome no. 11 contains ten genes (YKL225W, PAU16, YKL223W, YKL222C, MCH2, FRE2, COS9, SRY1, JEN1, URA1) and no. 12 contains fifteen genes (YHL050C, YKL050W-A, YHL049C, YHL048C-A, COS8, YHLComega1, ARN2, YHL046W-A, PAU13, YHL045W, YHL044W, ECM34, YHL042W, YHL041W, ARN1). We assumed that the loss of these genes resulted in the ethanol-tolerant phenotype and expect that this genome reconstruction method will be a feasible new alternative for strain improvement.

• Journal of Microbiology and Biotechnology
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• v.27 no.9
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• pp.1692-1700
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• 2017

Ralstonia solanacearum causes bacterial wilt in a wide variety of host plant species and produces a melanin-like blackish-brown pigment in stationary phase when grown in minimal medium supplemented with tyrosine. To study melanin production regulation in R. solanacearum, five mutants exhibiting overproduction of melanin-like pigments were selected from a transposon (Tn) insertion mutant library of R. solanacearum SL341. Most of the mutants, except one (SL341T), were not complemented by the original gene or overproduced melanins. SL341T showed Tn insertion in a gene containing a conserved domain of eukaryotic transcription factor. The gene was annotated as a hypothetical protein, given its weak similarity to any known proteins. Upon complementation with its original gene, the mutant strains reverted to their wild-type phenotype. SL341T produced 3-folds more melanin at 72 h post-incubation compared with wild-type SL341 when grown in minimal medium supplemented with tyrosine. The chemical analysis of SL341T cultural filtrate revealed the accumulation of a higher amount of homogentisate, a major precursor of pyomelanin, and a lower amount of dihydroxyphenylalanine, an intermediate of eumelanin, compared with SL341. The expression study showed a relatively higher expression of hppD (encoding hydroxyphenylpyruvate dioxygenase) and lower expression of hmgA (encoding homogentisate dioxygenase) and nagL (encoding maleylacetoacetate isomerase) in SL341T than in SL341. SL341 showed a significantly higher expression of tyrosinase gene compared with SL341T at 48 h post-incubation. These results indicated that R. solanacearum produced both pyomelanin and eumelanin, and the novel hypothetical protein is involved in the negative regulation of melanin production.

• Proceedings of the Korean Society of Soil and Groundwater Environment Conference
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• 2001.02a
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• pp.14-28
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• 2001

As basic study for purpose bioremedation in oil-contaminated environment, Primarily, we isolated biosurfactant producer- strains utilized of oil-agar plate, and measured surface tension and emulsifying activity. We investigated in oil-contaminated soil and sea water. In this laboratory, Pseudomonas sp. EL-012S strain isolated from oil-contaminated soil was able to product novel biosurfactant under the optimal culture condition. Its condition was n-hexadecane 2.0%, NH$_4$NO$_3$0.4%, Na$_2$HPO$_4$0.6%, KH$_2$PO$_4$0.4%, MgSO$_4$.7$H_2O$ 0.02%, CaCl$_2$.2$H_2O$ 0.001%, FeSO.7$H_2O$ 0.001%, initial pH 7.0 and aeration at 3$0^{\circ}C$, respectively. This biosurfactant was produced in both late-exponential and early-stationary phase. The biosurfactant from Pseudomonas sp. EL-012S was composed of carbohydrate, lipid and protein. The purified-biosurfactant was examined two (biosurfactant type I, II) with the silica gel G60 column chromatography and the purified biosurfactant confirmed thin layer chromatography, high performed liquid chromatography and gas chromatography. The biosurfactant type I involved in carbohydrate-lipid-protein characteristics lowered surface tension of water to 27dyne/cm and interfacial tension 4.5dyne/cm aginst to n-hexadecane and the biosurfactant type B involved in carbohydrate lipid characteristics lowered surface tension of water to 30dyne/cm and interfacial tension 8dyne/cm against to n-hexadecane. Specially type I had the properties such as strong emulsifying activity, emulsion stability, pH-stability, thermo-stability, high cleaning activity and forming ability.

• Mycobiology
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• v.45 no.4
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• pp.401-408
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• 2017

Colletotrichum gloeosporioides is an economically important fungal pathogen causing substantial yield losses indifferent host plants. To understand the genetic diversity and molecular epidemiology of this fungus, we have developed a novel, high-resolution multi-locus microsatellite typing (MLMT) method. Bioinformatic analysis of C. gloeosporioides unannotated genome sequence yielded eight potential microsatellite loci, of which five, CG1 $(GT)_n$, CG2 $(GT1)_n$, CG3 $(TC)_n$, CG4 $(CT)_n$, and CG5 $(CT1)_n$ were selected for further study based on their universal amplification potential, reproducibility, and repeat number polymorphism. The selected microsatellites were used to analyze 31 strains of C. gloeosporioides isolated from 20 different host plants from India. All microsatellite loci were found to be polymorphic, and the approximate fragment sizes of microsatellite loci CG1, CG2, CG3, CG4, and CG5 were in ranges of 213-241, 197-227, 231-265, 209-275, and 132-188, respectively. Among the 31 isolates, 55 different genotypes were identified. The Simpson's index of diversity (D) values for the individual locus ranged from 0.79 to 0.92, with the D value of all combined five microsatellite loci being 0.99. Microsatellite data analysis revealed that isolates from Ocimum sanctum, Capsicum annuum (chili pepper), and Mangifera indica (mango) formed distinct clusters, therefore exhibited some level of correlation between certain genotypes and host. The developed MLMT method would be a powerful tool for studying the genetic diversity and any possible genotype-host correlation in C. gloeosporioides.

• Mycobiology
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• v.48 no.5
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• pp.383-391
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• 2020

Use of nanoparticles (NPs) in several commercial products has led to emergence of novel contaminants of air, soil and water bodies. The NPs may exhibit greater ecotoxicity due to nano-scale dependent properties over their bulk counterparts. The present investigation explores the effect of in vitro supplementation of TiO₂, silica and silver NPs on radial growth and ultrastructural changes in the hyphae and spores of two mushroom genera, Ganoderma lucidum and Volvariella volvaceae. A concentration dependent decrease in radial growth on NP amended potato dextrose agar medium was recorded. However, in comparison to control, there was decrease in radial diameter on supplementation with TiO₂ NPs while an increase was recorded for silica and silver NPs amendments as compared to their bulk salts at same concentrations after 48 h of incubation. Optical microscopy studies showed decrease in the number of spores while increase in spore diameter and thinning of hyphal diameter on NPs supplementation. Scanning electron microscopy analysis of fungal growth showed presence of deflated and oblong spores in two fruiting strains of Ganoderma while Volvariella exhibited decreased sporulation. Further, hyphal thinning and branching was recorded in response to NP amendments in both the test mushrooms. Enhancement of protein content was observed on NP compared to bulk supplementation for all cultures, concentrations and hours of incubation except for TiO₂ NPs. Likewise, bulk and NP supplementations (at 100 mg L^-1) resulted in enhanced laccase activity with occurrence of laccase specific protein bands on SDS-PAGE analysis.

• The Plant Pathology Journal
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• v.33 no.4
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• pp.362-369
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• 2017

White root rot disease, caused by the pathogen Rosellinia necatrix, is one of the world's most devastating plant fungal diseases and affects several commercially important species of fruit trees and crops. Recent global outbreaks of R. necatrix and advances in molecular techniques have both increased interest in this pathogen. However, the lack of information regarding the genomic structure and transcriptome of R. necatrix has been a barrier to the progress of functional genomic research and the control of this harmful pathogen. Here, we identified 10,616 novel full-length transcripts from the filamentous hyphal tissue of R. necatrix (KACC 40445 strain) using PacBio single-molecule sequencing technology. After annotation of the unigene sets, we selected 14 cell cycle-related genes, which are likely either positively or negatively involved in hyphal growth by cell cycle control. The expression of the selected genes was further compared between two strains that displayed different growth rates on nutritional media. Furthermore, we predicted pathogen-related effector genes and cell wall-degrading enzymes from the annotated gene sets. These results provide the most comprehensive transcriptomal resources for R. necatrix, and could facilitate functional genomics and further analyses of this important phytopathogen.

• Journal of Veterinary Science
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• v.22 no.5
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• pp.59.1-59.13
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• 2021

Background: Antimicrobial peptides (AMPs) have been identified as promising compounds for consideration as novel antimicrobial agents. Objectives: This study analyzed the efficacy of cecropin B against Haemophilus parasuis isolates through scanning electron microscopy (SEM) and atomic force microscopy (AFM) experiments. Results: Cecropin B exhibited broad inhibition activity against 15 standard Haemophilus parasuis (HPS) strains and 5 of the clinical isolates had minimum inhibition concentrations (MICs) ranging from 2 to 16 ㎍/mL. Microelectrophoresis and hexadecane adsorption assays indicated that the more hydrophobic and the higher the isoelectric point (IEP) of the strain, the more sensitive it was to cecropin B. Through SEM, multiple blisters of various shapes and dents on the cell surface were observed. Protrusions and leakage were detected by AFM. Conclusions: Based on the results, cecropin B could inhibit HPS via a pore-forming mechanism by interacting with the cytoplasmic membrane of bacteria. Moreover, as cecropin B concentration increased, the bacteria membrane was more seriously damaged. Thus, cecropin B could be developed as an effective anti-HPS agent for use in clinical applications.

• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.610-620
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• 2021

There has been increasing interest in the head and neck squamous cell carcinoma (HNSCC) that is caused by high-risk human papillomavirus (HR-HPV) and has posed a significant challenge to Otolaryngologists. A rapid, sensitive, and reliable method is required for the detection of HR-HPV in clinical specimens to prevent and treat HPV-induced diseases. In this study, a multiple cross-linking spiral amplification (MCLSA) assay was developed for the visual detection of HPV-16. In the MCLSA assay, samples were incubated under optimized conditions at 62℃ for 45 min, and after mixing with the SYBR Green I (SGI) dye, the positive amplicons showed bright green fluorescence while the negative amplicons exhibited no obvious change. The specificity test revealed that the developed MCLSA technique had high specificity and could effectively distinguish all five HPV-16 strains from other pathogenic microorganisms. In terms of analytical sensitivity, the limit of detection (LoD) of MCLSA assay was approximately 5.4 × 101 copies/tube, which was 10-fold more sensitive than loop-mediated isothermal amplification (LAMP) and RT-PCR. The detection results of laryngeal cancer specimens collected from 46 patients with suspected HPV infection in the Liaoning region demonstrated that the positive detection rates of MCLSA and hybridized capture 2 kit were 32.61% (15/46). The true positive rate of the MCLSA assay was higher than that of RT-PCR (100% vs. 93.33%) and LAMP (100% vs. 86.67%). Therefore, the MCLSA assay developed in the present study could be a potentially useful tool for the point-of-care (PoC) diagnosis of HR-HPV, especially in resource-limited countries.

• Genomics & Informatics
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• v.16 no.4
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• pp.26.1-26.12
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• 2018

Shigella spp. constitutes some of the key pathogens responsible for the global burden of diarrhoeal disease. With over 164 million reported cases per annum, shigellosis accounts for 1.1 million deaths each year. Majority of these cases occur among the children of the developing nations and the emergence of multi-drug resistance Shigella strains in clinical isolates demands the development of better/new drugs against this pathogen. The genome of Shigella flexneri was extensively analyzed and found 4,362 proteins among which the functions of 674 proteins, termed as hypothetical proteins (HPs) had not been previously elucidated. Amino acid sequences of all these 674 HPs were studied and the functions of a total of 39 HPs have been assigned with high level of confidence. Here we have utilized a combination of the latest versions of databases to assign the precise function of HPs for which no experimental information is available. These HPs were found to belong to various classes of proteins such as enzymes, binding proteins, signal transducers, lipoprotein, transporters, virulence and other proteins. Evaluation of the performance of the various computational tools conducted using receiver operating characteristic curve analysis and a resoundingly high average accuracy of 93.6% were obtained. Our comprehensive analysis will help to gain greater understanding for the development of many novel potential therapeutic interventions to defeat Shigella infection.