• Korean Journal of Microbiology
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• v.42 no.4
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• pp.319-325
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• 2006

[ $\beta$ ]-Glucans (AG) were prepared from Agaricus blazei cultured in the medium fortified with the roots of Pueraria spp. by repeated extraction with hot water, gel filtration chromatography and DEAE ion exchange chromatography. Oligosaccharides (AO) were derived from the hydrolysis of AG by an endo-$\beta$-(1$\rightarrow$6)-glucanase from Bacillus megaterium. The anti-HT-29 human colon cancer activity of AG or AO was investigated using MTT assay, apoptosis assay, cell cycle analysis, and cDNA microairay. AG and AO both inhibited proliferation and growth of HT-29 cells, and stimulated apoptosis of the cells in a dose-dependent manner. In cell cycle analysis, treating HT-29 cells with AG or AO resulted in the increase of cells in the G0 (sub-G1) and G1 phase. Especially, AO was more effective in inducing G0/G1 cell cycle arrest than AG. To screen the genes involved in the increase of apoptosis, the gene expression profile of the HT-29 cells treated with AO was examined by cDNA microarray. While several genes involved in cell cycle progression (CCND2 and CDK2) were down-regulated, many genes involved in apoptosis (TNFSF9, TNFRSF9, FADD, CASP8, BAD, CRADD, CASP9 etc), cell cycle inhibitor (CDKN2A), immune response (IL6, IL18, IL6R etc), and tumor suppressor (CEACAM1, TP53BP2, IRF1, and PHB) were up-regulated. These results suggest that AO could inhibit the proliferation and growth of HT-29 cells by G0/G1 cell cycle arrest and induction of apoptosis.

• Korean Journal of Microbiology
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• v.51 no.1
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• pp.39-47
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• 2015

Secondary metabolism in actinomycetes has been known to be controlled by a small molecule, ${\gamma}$-butyrolactone autoregulator, the binding of which to each corresponding receptor leads to the regulation of the transcriptional expression of the secondary metabolites. We expected that expression of an autoregulator receptor or a pleiotropic regulator in a non-host was to be gained insight of effective production of new metabolic materials. In order to study the function of the receptor protein (seaR), which is isolated from Saccharopolyspora erythraea, we introduced the seaR gene to Streptomyces coelicolor A3(2) as host strains. An effective transformation procedure for S. coelicolor A3(2) was established based on transconjugation by Escherichia coli ET12567/pUZ8002 with a ${\varphi}C31$-derived integration vector, pSET152, which contained int, oriT, attP and $ermEp^*$ (erythromycin promotor). Therefore, the pEV615 was introduced into S. coelicolor A3(2) by conjugation and integrated at the attB locus in the chromosome of the recipients by the ${\varphi}C31$ integrase (int) function. Exconjugant of S. coelicolor A3(2) containing the seaR gene was confirmed by PCR and transcriptional expression of the seaR gene in the transformant was analyzed by RT-PCR. In case of S. coelicolor A3(2), a phenotype microarray was used to analyze the phenotype of transformant compared with wild type by seaR expression. After that, in order to confirm the accuracy of the results obtained from the phenotype microarray, an antimicrobial susceptibility test was carried out. This test indicated that sensitivity of the transformant was higher than wild type in tetracycline case. These results indicated that some biosynthesis genes or resistance genes for tetracycline biosynthesis in transformant might be repressed by seaR expression. Therefore, subsequent experiments, analysis of transcriptional pattern of genes for tetracycline production or resistance, are needed to confirm whether biosynthesis genes or resistance genes for tetracycline are repressed or not.

• Korean Journal of Microbiology
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• v.51 no.1
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• pp.7-13
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• 2015

Hydrotrope-combined copper (HCC) is a copper ($Cu^{2+}$)-based algicide, which is combined with a hydrotrope that keeps copper ion in solution to improve performance. This study assessed the growth inhibition effect of HCC against Microcystis aeruginosa which is one of the most common toxic cyanobacterium in eutrophic freshwater environment. Various HCC doses, ranging from 5.5 to $550{\mu}g/L$ as $Cu^{2+}$, were applied to either BG-11 or 1/4 diluted medium with low- or high-inoculum density of M. aeruginosa. Growth inhibition was monitored based on a decrease in chlorophyll-a content in culture medium during the incubation. Results showed that HCC significantly inhibited the growth of M. aeruginosa in a dose-dependent manner. In case of 1/4 diluted BG-11 medium, HCC dose as low as $5.5{\mu}g$ $Cu^{2+}/L$ completely inhibited the production of chlorophyll-a by M. aeruginosa. It was found that HCC did not induce any significant release of microcystin-LR from M. aeruginosa. Acute toxicity of HCC was tested using Daphnia magna, and the 24-h $EC_{50}$ value was 0.30 mg/L as $Cu^{2+}$ which was much higher than the actual inhibition dose. Ames test was performed using Salmonella enterica serovar Typhimurium TA100, and HCC showed no increase in the number of revertant colonies. The result suggested that HCC does not have any mutagenic potential in the aquatic environment. In addition, no genotoxic effect of HCC was also confirmed based on the SOS ChromoTest using Escherichia coli PQ37. Therefore, HCC could be used as a relatively safe and effective pre- and post-treatment agent to control hazardous algal blooming in aquatic environments.

• Korean Journal of Microbiology
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• v.51 no.2
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• pp.97-107
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• 2015

Wetlands constitute a transitional zone between terrestrial and aquatic ecosystems and have unique characteristics such as frequent inundation, inflow of nutrients from terrestrial ecosystems, presence of plants adapted to grow in water, and soil that is occasionally oxygen deficient due to saturation. These characteristics and the presence of vegetation determine physical and chemical properties that affect decomposition rates of organic matter (OM). Decomposition of OM is associated with activities of various extracellular enzymes (EE) produced by bacteria and fungi. Extracellular enzymes convert macromolecules to simple compounds such as labile organic carbon (C), nitrogen (N), phosphorus (P), and sulfur (S) that can be easily taken up by microbes and plants. Therefore, the enzymatic approach is helpful to understand the decomposition rates of OM and nutrient cycling in wetland soils. This paper reviews the physical and biogeochemical factors that regulate extracellular enzyme activities (EEa) in wetland soils, including those of ${\beta}$-glucosidase, ${\beta}$-N-acetylglucosaminidase, phosphatase, arylsulfatase, and phenol oxidase that decompose organic matter and release C, N, P, and S nutrients for microbial and plant growths. Effects of pH, water table, and particle size of OM on EEa were not significantly different among sites, whereas the influence of temperature on EEa varied depending on microbial acclimation to extreme temperatures. Addition of C, N, or P affected EEa differently depending on the nutrient state, C:N ratio, limiting factors, and types of enzymes of wetland soils. Substrate quality influenced EEa more significantly than did other factors. Also, drainage of wetland and increased temperature due to global climate change can stimulate phenol oxidase activity, and anthropogenic N deposition can enhance the hydrolytic EEa; these effects increase OM decomposition rates and emissions of $CO_2$ and $CH_4$ from wetland systems. The researches on the relationship between microbial structures and EE functions, and environmental factors controlling EEa can be helpful to manipulate wetland ecosystems for treating pollutants and to monitor wetland ecosystem services.

• Korean Journal of Microbiology
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• v.51 no.2
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• pp.126-132
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• 2015

Three different methods (simple washing of plastic and pulp sample, washing after direct heating of the diapers, and the heating after washing of plastic and pulp sample) were carried out to decrease total coliforms and heterotrophic plate count (HPC) in the diaper's plastic and pulp. Plastic and pulp samples were obtained from diaper by treatment with 10% $CaCl_2$ and 4% sea salt water, dilution with 1,000 ml tap water, and draining by using sieves. Three times washing was the most appropriate for the reduction of microorganisms in plastic and pulp. By three times washing, the number of total coliforms in the plastic and pulp samples showed 92.8% and 99.8% of decrease, respectively, and the number of HPC showed 97.3% of decrease in the plastic and 98.5% of decrease in the pulp. The washing after direct heating of the fecal contaminated diapers was not effective because HPC in the plastic and pulp samples were still detected about 2-3 log CFU/g in the plastic and 1-2 log CFU/g in the pulp, respectively, even after heating at $60^{\circ}C$, $80^{\circ}C$, $100^{\circ}C$ for 12 h. Meanwhile, total coliforms and HPC were completely sterilized at $80^{\circ}C$ for 4 h by heating after washing of plastic and pulp samples, suggesting that this method was the most appropriate method for the reduction of microorganisms in plastic and pulp obtained from fecal contaminated diapers.

• Korean Journal of Microbiology
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• v.43 no.3
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• pp.151-158
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• 2007

The possibility of inadvertent introduction of therapeutic gene expressing viral vectors has raised safety concerns about germ-line infection. Particularly, for indications such as prostate cancer and ovarian cancer, the proximity of the point of viral administration to organs of the reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus vector. To evaluate the safety of in vivo adenovirus mediated gene transfer, we explored the biodistribution, persistance and potential germ-line transmission of p53-expressing adenovirus (Ad-CMV-p53). Both male and female Balb/c mice were injected with $1{\times}10^9$ PFU of Ad-CMV-p53. The PCR analysis showed that there were detectable vector sequences in liver, kidney, spleen, seminal vesicle, epididymis, prostate, ovary, and uterus. The RT-PCR analysis for detecting inserted gene, p53 showed that Ad-CMV-p53 viral RNA were present in spleen, prostate and ovary. Direct injected male and female mice of adenovirus vector into testis and ovary were mated and their of offspring were evaluated for germ-line transmission of the adenoviral vector. The PCR and RT-PCR analysis showed no evidence of germline transmission, although vector sequences were detected in DNA extracted from gonadal tissues. Real-time PCR result confirmed a significant decrease of adenovirus in gonad tissues 1 week after injection. We have also analysed the cell specific localization of viral DNA in gonad tissues by using in-situ PCR. Positive signals were detected in interstitial tissue but not in seminiferous tubule in sperm. In the case of ovary, adenovirus signal were localized to the stromal tissue, but no follicular signals were observed. Together, these data provide strong evidence that the risk of the Inadvertent germ-line transmission of vector sequences following intraperitoneal or direct injection into genito-urinary system of adenovirus is extremely low.

• Korean Journal of Microbiology
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• v.48 no.3
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• pp.180-186
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• 2012

Pseudomonas aeruginosa, a Gram-negative bacterium, is an ubiquitous and opportunistic human pathogen, which expresses many virulence factors through quorum sensing (QS) regulation. QscR, one of the QS signal receptors of P. aeruginosa, has unique features that make it possible to distinguish QscR from other QS receptors. In the present study, we focused on amino acid residues responsible for such a broad signal specificity of QscR. Thus we constructed mutant QscRs: $QscR_{T72I}$, $QscR_{R132M}$, and $QscR_{T140I}$ by substituting $72^{nd}$ threonine, $132^{nd}$ arginine, and $140^{th}$ threonine residues with isoleucine, methionine, and isoleucine, respectively by site-directed mutagenesis. When we examined the activity of these mutant QscRs, $QscR_{R132M}$ failed to respond to N-3-oxododecanoyl homoserine lactone (3OC12-HSL), but $QscR_{T72I}$ and $QscR_{T140I}$ remained the ability to respond to 3OC12-HSL despite much reduction of the sensitivity. When we treated a variety of acyl-HSLs with different structure, $QscR_{T72I}$ and $QscR_{T140I}$ showed better responsiveness to N-decanoyl HSL (C10-HSL) or N-dodecanoyl HSL (C12-HSL) that has no oxo-moiety at $3^{rd}$ carbon of acyl group than to 3OC12-HSL, and $QscR_{R132M}$ showed no responsiveness to any acyl-HSLs tested here. In addition, $QscR_{T72I}$ and $QscR_{T140I}$ were inhibited by 5f, a QscR inhibitor as similarly as wild type QscR was. These results suggest that while the $130^{th}$ arginine is crucial in both activity and acyl-HSL binding of QscR, the $72^{nd}$ and $140^{th}$ threonines are important in the activity, but they are little responsible for the discrimination of acyl-HSLs or competitive inhibitor.

• Korean Journal of Materials Research
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• v.6 no.7
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• pp.700-708
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• 1996

Electroless plating technique was utilized to flip chip bonding to improve surface mount characteristics. Each step of plating procedure was studied in terms pf pH, plating temperature and plating time. Al patterned 4 inch Si wafers were used as substrstes and zincate was used as an activation solution. Heat treatment was carried out for all the specimens in the temperature range from room temperature to $400^{\circ}C$ for $30^{\circ}C$ minutes in a vacuum furnace. Homogeneous distribution of Zn particles of size was obtained by the zincate treatment with pH 13 ~ 13.5, solution concentration of 15 ~ 25% at room temperature. The plating rates for both Ni-P and Au electroless plating steps increased with increasing the plating temperature and pH. The main crystallization planes of the plated Au were found to be (111) a pH 7 and (200) and (111) at pH 9 independent of the annealing temperature.

• Korean Journal of Materials Research
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• v.10 no.8
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• pp.569-574
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• 2000

To improve the ductility of $Al_3Hf$ and $Al_3Ta$ intermetallic compounds, which are the potential temperature structural materials, the mechanical alloying behaviour and the effect of ternary additions on the $Ll_2$ phase formation were investigated. During the mechanical alloying by the SPEX mill, the $Ll_2$ $Al_3Hf$ intermetallic compound was formed after 6 hours of milling in AL-25%Hf system. In AL-25%Ta system, however, only the $D0_{22}$ $Al_3Ta$ intermetallic compound was formed until 30 hours of milling and the $Ll_2$ phase was not observed. In AL-12.5%M-25%Ta(M=Cu, Zn, Mn, Fe, Ni) systems, the additions of Cu and Zn had no effect on the $D0_{22}$ structure of the binary $Al_3Hf$ and the additions of Mn, Fe and Ni produced the amorphous phase. Therefore it was considered that these ternary additions could not overcome the energy difference between $Ll_2$ and $D0_{22}$ structures in the $Al_3Hf$ intermetallic compound. In AL-12.5%M-25%Hf(M=Cu, Zn, Mn, Fe, Ni)systems, the additions of Cu and Zn did not affect the $Ll_2$ structure of the binary $Al_3Hf$ but the additions of oMn, Fe and Ni produced the amorphous phase as they did in AL-12.5%M-25%Ta systems. Therefore, it was considered that the Ni, Mn and Fe additions promote the formation of amorphous phase in $Al_3X$ intermetallic compounds.

• Korean Journal of Microbiology
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• v.45 no.3
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• pp.286-290
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• 2009

The transcription of mRNA of avian influenza virus is regulated temporally during infection. Therefore, the measurement of transcript level in host cells should be performed before viral release from host cells because errors can occur in the analysis of the transcript levels if the viruses released from the infected cells re-infect cells. In this study, the timing of viral release was determined by measuring the level of viral RNA from viruses released from H9N2-infected chicken fibroblast cell line UMNSAH/DF-1 by semi-quantitative RT-PCR. The viral genomic RNA was isolated together with mouse total RNA which was added to the collected medium as carrier to monitor the viral RNA recovery and to use its GAPDH as an internal control for normalizing reverse transcription reaction as well as PCR reaction. It was found that viral release of H9N2 in the chicken fibroblast cell line UMNSAH/DF-1 took between 16 and 20 h after infection. We measured all 8 viral mRNA levels. Of the 8 transcripts, 7 species of viral mRNAs (each encoding HA, NA, PB1, PB2, NP, M, NS, respectively) except PA mRNA showed robust amplification, indicating these mRNA can be used as targets for amplification to measure transcript levels. These results altogether suggest that the method in this study can be used for screening antiviral materials against viral RNA polymerase as a therapeutic target.