• Microbiology and Biotechnology Letters
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• v.30 no.3
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• pp.235-240
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• 2002

Streptomycetes is a group of Gram-positive soil bacteria that growas a branching vegetative mycelium leading to the formation of spores, and display a physiological differenti-ation related to the synthesis of many secondary metabolites including antibiotics. Their complex life cycle and multicellular differentiation require various levels of regulation and types of signal transduction systems including eukaryotic-type serine/threonine protein kinases and prokaryotic-type histidine/aspartic acid protein kinases. Akt kinase that was found in cells is a sorine/threonine kinase controlling signal pathway for multi-tude of important cellular events. The activation or inactivation of Akt kinase in the cell is one of the critical regulatory points to deliver cell proliferation, differentiation, survival or apoptosis signal. To find the regula-tory protein homologous to Akt in Streptomyces, the fluorescien-labeled synthetic peptide (FITC-TRRSR-TESIT) was designed from the consensus sequence of target proteins for Akt kinase. From the difference of the mobility between the nonphosphorylated and phosphorylated synthetic peptides on Agarose gel electro-phoresis, the Akt-phosphorylating activity was monitored. The cell-free extract prepared from Streptomyces griseus IFO 13350 and the Akt homologous protein was purified by ammonium sulfate fractionation and many steps of column chromatographies such as, DEAE-Sepharose, Mono Q, Resource Phenyl-Soporose and Gel permeation column chromatographies. As a result, the protein phosphorylating the fluorescien-labeled Akt substrate was identified and it's molecular weight was estimated as 39 kDa on SDS-PAGE.

• Microbiology and Biotechnology Letters
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• v.39 no.2
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• pp.104-110
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• 2011

Actinomycetes, Gram positive soil bacteria, are valuable microorganisms which produce useful secondary metabolites including antibiotics, antiparasitic substances, anti-cancer drugs, and immunosuppressants. Although a major family of actinomycetes, known as streptomycetes, has been intensively investigated at the molecular level for several decades, a potentially valuable and only recently isolated non-streptomycetes rare actinomycetes (NSRA) family has been poorly characterized due to lack of proper genetic manipulation systems. Here we report that a PCR-based genome screening strategy was performed with approximately 180 independently isolated actinomycetes strains to isolate potentially valuable NSRA strains. Thanks to this simple PCR-based genome screening strategy we were able to identify only seven NSRA strains, followed by 16S rRNA sequencing for confirmation. Through further bioassays, one potentially valuable NSRA strain (tentatively named Nocardiopsis species MMBL010) was identified which possessed both antifungal and antibacterial activities, along with the presence of polyketide synthase and non-ribosomal peptide synthase genes. Moreover, Nocardiopsis species MMBL010, which was intrinsically recalcitrant to genetic manipulation, was successfully transformed via E. coli-driven conjugation. These results suggest that PCR-based genome screening, followed by the establishment of an E. coli-driven conjugation system, is an efficient strategy to maximize potentially valuable compounds and their biosynthetic genes from NSRA strains isolated from various environments.

• Microbiology and Biotechnology Letters
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• v.41 no.4
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• pp.433-441
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• 2013

In this study, the anti-oxidative, anti-inflammatory, anti-melanogenic activities of Endlicheria anomala (Nees) Mez methanol extract (EAME) were evaluated by use of in vitro assays and cell culture model systems. The results revealed that EAME scavenges various radicals such as 1,1-diphenyl-2-picryl hydrazyl hydrogen peroxide induced reactive oxygen species, and lipopolysaccharide induced nitric oxide. Furthermore, EAME induced the expression of anti-oxidative enzymes such as heme oxygenase 1, thioredoxin reductase 1, NAD(P)H dehydrogenase 1, and their upstream transcription factor, nuclear factor-E2-related factor 2. Moreover, EAME inhibited in vitro DOPA oxidation and 3-isobutyl-1-methylxanthine induced melanogenesis in B16F10 cells. Its anti-melanogenic activity will have originated from the inhibition of tyrosinase enzyme activity and melanogenesis related protein expression. Taken together, these results provide the important new insight that E. anomala possesses various biological activities such as anti-oxidative, anti-inflammatory, and anti-melanogenic. Therefore, it might be utilized as a promising material in the fields of nutraceuticals and cosmetics.

• Journal of Microbiology and Biotechnology
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• v.23 no.11
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• pp.1509-1518
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• 2013

An agar-degrading bacterium, designated as strain $G7^T$, was isolated from a coastal seawater sample from Gaya Island (Gayado in Korean), Republic of Korea. The isolated strain $G7^T$ is gram-negative, rod shaped, aerobic, non-motile, and non-pigmented. A similarity search based on its 16S rRNA gene sequence revealed that it shares 95.5%, 90.6%, and 90.0% similarity with the 16S rRNA gene sequences of Catenovulum agarivorans $YM01^T$, Algicola sagamiensis, and Bowmanella pacifica W3-$3A^T$, respectively. Phylogenetic analyses demonstrated that strain $G7^T$ formed a distinct monophyletic clade closely related to species of the family Alteromonadaceae in the Alteromonas-like Gammaproteobacteria. The G+C content of strain $G7^T$ was 41.12 mol%. The DNA-DNA hybridization value between strain $G7^T$ and the phylogenetically closest strain $YM01^T$ was 19.63%. The genomes of $G7^T$ and $YM01^T$ had an average ANIb value of 70.00%. The predominant isoprenoid quinone of this particular strain was ubiquinone-8, whereas that of C. agarivorans $YM01^T$ was menaquinone-7. The major fatty acids of strain $G7^T$ were Iso-$C_{15:0}$ (41.47%), Anteiso-$C_{15:0}$ (22.99%), and $C_{16:1}{\omega}7c/iso-C_{15:0}2-OH$ (8.85%), which were quite different from those of $YM01^T$. Comparison of the phenotypic characteristics related to carbon utilization, enzyme production, and susceptibility to antibiotics also demonstrated that strain $G7^T$ is distinct from C. agarivorans $YM01^T$. Based on its phenotypic, chemotaxonomic, and phylogenetic distinctiveness, strain $G7^T$ was considered a novel genus and species in the Gammaproteobacteria, for which the name Gayadomonas joobiniege gen. nov. sp. nov. (ATCC BAA-2321 = $DSM25250^T=KCTC23721^T$) is proposed.

• Journal of Life Science
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• v.20 no.8
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• pp.1281-1286
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• 2010

Gaucher disease (GD) is caused by glucocerebrosidase functional deficiency and the most prevalent lysosomal storage disorder (LSD), with an incidence of about 1 in 20,000 new births. Resveratrol, one kind of phytoalexin, is a produced naturally by several plants and has anti-tumor, anti-aging, anti-inflammatory and neuro-protective effects. In this paper we provide the cellular protective effect of resveratrol in both type I and type II Gaucher disease cells. Resveratrol treatment did not show any significant change in the p21 and p53 mRNA expression level, however expression level of the p21 protein, a cell cycle arrest factor, shows significant increment in both types of Gaucher disease cells. These cell cycle arrest patterns were confirmed by both MTT assay measurement and microscopy detection. In comparison, expression level of poly ADP ribose polymerase (PARP), an apoptosis indicator protein, was significantly decreased in both type I and II Gaucher disease cells after treatment with resveratrol. This result indicates that resveratrol relievescellular apoptotic stress fromtype I and II Gaucher disease cells. Therefore, we demonstrate that resveratrol inhibits cell proliferation via p21 activity and activates cellular repair systems for Gaucher disease cells. Our results provide at least one of the molecular mechanisms of Gaucher disease and may allow the verification of potential drug targets for therapeutic trials.

• Journal of Marine Bioscience and Biotechnology
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• v.2 no.1
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• pp.48-54
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• 2007

Bacillus polyfermenticus SCD (B. polyfermenticus SCD) has been appropriately used for the therapy of long-term intestinal disorders, because live strains in the form of active endospores can successfully reach the target intestine in humans. B. polyfermenticus SCD produces the most antibiotic-related materials. In the present study, B. polyfermenticus SCD was fermented with soybeans (BPFS) and its probiotic properties were investigated. B. polyfermenticus SCD and BPFS showed a broad spectrum of antimicrobial activity against pathogenic Gram-positive (Streptococcus parauberis, Streptococcus iniae, Lactococcus garviae) and Gram-negative (Flexibacter tractuosus, Vibrio harveyi, Vibrio vulnificus, Vibrio ordalii) bacteria and moulds (Aspergillus niger, Aspergillus oryzae). Sebastes schlegeli were used to examine survival rate and cleanup action by BPFS. Bacterial infection resulted in a mortality of up to 99% in the commercial fodder fed groups. BPFS both enhanced the growth rate of fry by improving their appetite and had cleaned by decreased eutrophication. Therefore, it seems appropriate that BPFS should be developed as an antibiotic replacement, favorable fodder additive, and antifungal material in fish farming systems.

• Journal of Dairy Science and Biotechnology
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• v.27 no.1
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• pp.1-12
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• 2009

In 2008, baby formula containing melamine was found to be responsible for a large outbreak of renal failure in infants in China. A total of 294,000 infants were hospitalized, and at least 6 babies died due to ingestion of the tainted formula. Melamine contains high levels of nitrogen (>60%), which is used as an indicator of protein content. Therefore, high levels of melamine in infant formula were thought to be the result of deliberate contamination m an attempt to increase its apparent protein content. Following inspections by China's national inspection agency, assorted products from at least 22 dairy manufacturers across China were found to have varied levels of melamine (range: 0.096196.61 mg/kg). Melamine co-exposure with cyanuric acid can induce acute melamine-cyanurate crystal nephropathy, which can lead to renal failure at much lower doses than if either compound were ingested alone. However, currently, there are very few data on melamine analogues other than cyanuric acid. At an expert meeting of the WHO and FAO held to review toxicological aspects of melamine and cyanuric acid on December 14, 2008, a new tolerable daily intake (TDI) of melamine was established that could be applied to the entire population, including infants. Therefore, a risk assessment of the various theoretical melamine contamination levels in infant formula and selected representative foods (other than infant formula and sole-source nutrition products) is urgently needed for Korean babies and children up to 7 years of age. Although the undetectable level regulation for infant formula may be low enough to guarantee the safety of babies under the age of 1 year (including premature babies), the melamine standard of 2.5 ppm for foods other than baby formula could be insufficient to protect the 95th percentile population aged 1~2 years because of this demographic's high consumption of milk, yogurt, and soy milk (hazard index = 1.79). Because TDIs are chronic values intended to protect an individual over his/her lifetime, occasional modest ingestion in excess of the TDI is not likely to be a health concern. However, children aged 1~2 years may have renal systems that are comparatively more sensitive to the crystallization of melamine and its analogues. Therefore, governmental jurisdictions may need to practice more prudent management of food items that could raise the melamine exposure for this population.

• Proceedings of the Korean Society of Plant Biotechnology Conference
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• 2005.11a
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• pp.71-80
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• 2005

Effects of low temperature ($8^{\circ}C$) on the hydraulic conductivity of young roots of a chilling-sensitive (cucumber; Cucumis sativus L.) and a chilling-resistant (figleaf gourd; Cucurbita ficifolia Bouche) crop have been measured at the levels of whole root systems (root hydraulic conductivity, $Lp_r$) and of individual cortical cells (cell hydraulic conductivity, Lp). In figleaf gourd, there was a reduction only in hydrostatic $Lp_r$ but not in osmotic $Lp_r$ suggesting that the activity of water channels was not much affected by low root temperature (LRT)treatment in this species. Changes in cell Lp in response to chilling and recovery were similar asroot level, although they were more intense at the root level. Roots of figleaf gourd recovered better from LRT treatment than those of cucumber. In figleaf gourd, recovery (both at the root and cell level) often resulted in Lp and $Lp_r$ values which were even bigger than the original, i.e. there was an overshoot in hydraulic conductivity. These effects were larger forosmotic (representing the cell-to-cell passage of water) than for hydrostatic $Lp_r$. After a short term (1 d) exposure to $8\;^{\circ}C$ followed by 1 d at $20\;^{\circ}C$, hydrostatic $Lp_r$ of cucumber nearly recovered and that of figleaf gourd still remained higher due to the overshoot. On the contrary, osmotic $Lp_r$ and cell Lp in both species remained high by a factor of 3 as compared to the control, possibly due to an increased activity of water channels. After pre-conditioning of roots at LRT, increased hydraulic conductivitywas completely inhibited by $HgCl_2$ at both the root and cell levels. Different from figleaf gourd, recovery from chilling was not complete in cucumber after longer exposure to LRT. It is concluded that at LRT, both changes in the activity of aquaporins and alterations of root anatomy determine the water uptake in both species. To better understand the aquaporin function in plants under various stress conditions, we examined the transgenic Arabidopsisand tobacco plants that constitutively overexpress ArabidopsisPIP1;4 or PIP2;5 under various abiotic stress conditions. No significant differences in growth rates were found between the transgenic and wild-type plants under favorable growth conditions. By contrast, overexpression of PIP1;4 or PIP2;5 had a negative effect on seed germination and seedling growth under drought stress, whereas it had a positive effect under cold stress and no effect under salt stress. Measurement of water transport by cell pressure probe revealed that these observed phenotypes under different stress conditions were closely correlated with the ability of water transport by each aquaporin in the transgenic plants. Together, our results demonstrate that PIP-type aquaporins play roles in seed germination, seedling growth, and stress response of Arabidopsis and tobacco plants under various stress conditions, and emphasize the importance of a single aquaporin-mediated water transport in these cellular processes.

• Journal of Plant Biotechnology
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• v.39 no.3
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• pp.196-204
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• 2012

This study was carried out to develop reliable systems for somatic embryogenesis in oil palm tree (Elaeis guineensis Jacq.), and to verify the somaclonal variants by RAPD analysis. Embryogenic callus was induced successfully on modified half-strength MS medium containing $NaH_2PO_4{\cdot}2H_2O$ and casein. Embryogenic callus was further developed to somatic embryo mass (SEM), which is very hard and bonded tightly each other. Plantlets were proliferated when SEM was cultured on modified MS medium containing half strength $NH_4NO_3$, casein and L-ascorbic acid. Plantlets were transplanted into pots containing artificial soils. When RAPD analysis was conducted using randomly selected 95 in vitro plantlets and 19 random primers, somaclonal variation was detected using BNR35 primer. There was missing band around 1 kb in #22, #28, #35, and #77 plantlets. In addition, bands obtained from #28, #35, and #77 was much stronger than other normal bands. The blast results at NCBI revealed that somaclonal variation observed in this study was related to chloroplast genome of oil palm. The results also revealed that oil palm reproduction system through somatic embryogenesis is quite reliable and early detection of somaclonal variants seem to be possible at in vitro stage by RAPD analysis.

• Journal of Plant Biotechnology
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• v.40 no.3
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• pp.169-177
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• 2013

Recently, the number of people with diabetes is rapidly increasing, coupled with the fact that the insulin market is remarkably increasing. Therefore, molecular farming for plant-derived pharmaceutical protein production is reported as becoming more attractive than ever. In this study, we carried out experiments step by step for development of recombinant insulin constructs, which were transformed into E. coli system, in vitro transcription and translation system, and tobacco cells. At first, recombinant proinsulin protein was successfully produced in in vitro transcription and translation system with wheat germ extract. After which, recombinant construct of prominiinsulin encoded a fusion protein of 7.8 kDa with trypsin cleavage sites at N terminus and C terminus of minimized C-peptide was tried to in vitro expression using E.coli culture. After purification with His-tag column, the resulting recombinant prominiinsulin protein was processed with trypsin, and then checked insulin biosynthesis by SDS-PAGE and western blot analysis with anti-insulin monoclonal antibody. The immunoreactive product of trypsin-treated miniinsulin was identical to the predicted insulin hexamer. The construct of 35S promoter-driven preprominiinsulin recombinant gene with signal peptide region for ER-targeting and red fluorescence protein gene [N terminus ${\rightarrow}$ tobacco E2 signal peptide ${\rightarrow}$ B-peptide (1-29 AA) ${\rightarrow}$ AAK ${\rightarrow}$ A-peptide (1-21 AA) ${\rightarrow}$ RR ${\rightarrow}$ His6 ${\rightarrow}$ KDEL ${\rightarrow}$ C terminus] was transformed into BY-2 tobacco cells. A polypeptide corresponding to the 38-kDa molecular mass predicted for fusion protein was detected in total protein profiles from transgenic BY-2 cells by western analysis. Therefore, this recombinant preprominiinsulin construct can be used for generation of transgenic tobacco plants producing therapeutic recombinant insulin.