• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1429-1433
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• 2006

Application of recombinant protein production and particularly their isotopic enrichment has stimulated development of a range of novel multidimensional heteronuclear NMR techniques. Peptides in most cases are amenable to assignment and structure determination without the need for isotopic labeling. However, there are many cases where the availability of $^{15}N$ and/or $^{13}C$ labeled peptides is useful to study the structure of peptides with more than 30 residues and the interaction between peptides and membrane. CRAMP (Cathelicidin-Related AntiMicrobial Peptide) was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin-derived antimicrobial peptides. CRAMP was successfully expressed as a GST-fused form in E. coli and purified using affinity chromatography and reverse-phase chromatography. The yield of the CRAMP was 1.5 mg/l 1. According to CD spectra, CRAMP adopted ${\alpha}$-helical conformation in membrane-mimetic environments. Isotope labeling of CRAMP is expected to make it possible to study the structure and dynamic properties of CRAMP in various membrane systems.

• Journal of Microbiology and Biotechnology
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• v.22 no.5
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• pp.600-606
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• 2012

Studies into the cell death program termed apoptosis have resulted in new information regarding how cells control and execute their own demise, including insights into the mechanism by which death-preventing factors can inhibit Bax-induced caspase activation. We investigated high temperature stress-induced cell death in Brassica rapa. Using a yeast functional screening from a Brassica rapa cDNA library, the BH5-127 EST clone encoding an apoptotic suppressor peptide was identified. However, a phylogenic tree showed that BH5-127 clusters within a clade containing SUMO-1 (Small Ubiquitin-like Modifier-1). BH5-127 was confirmed similar to have function to SUMO-1 as Fas suppression. Expression of BH5-127 showed that substantial suppression of cell death survived on SD-galactose-$Leu^-$-$Ura^-$ medium. The results suggest that BrSE ($\underline{B}$rassica rapa $\underline{S}$entrin $\underline{E}$ST, BH5-127) is one of the important regulatory proteins in programming cell death, especially in the seedling stage of Chinese cabbage.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.53-53
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• 2003

Calumenin was previously identified as a high affinity Ca$\^$2+/ binding protein in mouse cardiac sarcoplasmic reticulum (SR). For the present study, a 48 kDa skeletal homologue of calumenin was identified by sucrose-density gradient of rabbit skeletal SR membranes, concanavalin A treatment, 2D-gel electrophoresis, $\^$45/Ca$\^$2+/ overlay, Stains-all staining, and MALDI-TOF analysis. We attempted to clone the skeletal calumenin by RT-PCR based on mouse cardiac and human calumenin sequences. The deduced amino acid sequence (315 residues) of the skeletal calumenin showed high identity to mouse cardiac calumenin (90％). As seen in the cardiac calumenin, the deduced sequence contains a 19 amino acid N-terminal signal sequence and a HDEF C-terminal sequence, a putative retrieval signal to ER. Also, the skeletal calumenin contains one N-glycosylation site, three PKC phosphorylation sites, eight casein kinase 2 phosphorylation sites, and 6 EF-hand domains. GST-calumenin showed a conformational change and increased mobility in the presence of Ca$\^$2+/ in SDS-PAGE. Three calumenin interacting proteins (ryanodine receptor 1, glycogen phosphorylase, and phosphofructo kinase) were identified by pull-down assay with GST-calumenin and solubilized SR. All the interactions were Ca$\^$2+/dependent. The present results suggest that calumenin plays an important role in Ca$\^$2+/ homeostasis of muscle cells.

• Animal cells and systems
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• v.6 no.4
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• pp.305-309
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• 2002

Actinorhodin antibiotics produced by Streptomyces lividans TK24 are blue pigments with a weak antibiotic activity, derived from one acetyl-CoA and 15 malonyl-CoA units via a typical ployketide pathway. In an attempt to clone polyketide biosynthetic genes of S. lividans TK24, hybridizing fragments in the genomic DNA of S. lividans TK24 were detected by use of acn and act III polyketide synthase gene probes. Since typical aromatic polyketide bio-synthetic gene clusters are roughly 22-34 Kb long, we constructed in E. coli XL-Blue MR using the Streptomyces-E. coli bifunctional shuttle cosmid vector (pojn46). Then, about 5,000 individual E. coii colonies were thor-oughly screened with acrl-ORFI and actIII probes. From these cosmid libra-ries, 12 positive clones were identified. Restriction analysis and southern hybridization showed two polyketide biosynthetic gene clusters in this organism. These cosmid clones can be transformed into Streptomyces parvulus 12434 for expression test that identify product of actinorhodin biosynthetic genes by heterologous expression. Thus, heterologous expres-sion of a derivative compound of a actinorhodin biosynthetic intermediate was obtained in pKE2430. Expression of these compounds by the trans-formants was detected by photodiode array HPLC analysis of crude extracts.

• Animal cells and systems
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• v.9 no.4
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• pp.239-244
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• 2005

A cDNA clone for a wound- and pathogen-induced gene in Chinese cabbage (Brassica rapa subsp. pekinensis) was isolated and characterized. The cabbage gene, designated BrPR4, encodes a pathogenesis-related protein 4 (PR4) of 140 amino acids. The BrPR4 protein shows high similarity with wound-inducible antifungal proteins of tobacco, potato, barley, and wheat. The BrPR4 gene is locally induced by a nonhost pathogen, Pseudomonas syringae pv. tomato, that elicits a hypersensitive response in Chinese cabbage. Treatment of the cabbage leaves with benzothiadiazole (BTH), methyl jasmonate or ethephon showed that the BrPR4 gene expression is strongly induced by ethylene, but not by methyl jasmonate or BTH. The BrPR4 gene is also activated by wounding. Interestingly, however, the wound-inducible BrPR4 gene expression is repressed by salicylic acid or BTH, suggesting that there is cross-talk between salicylate-dependent and -independent signaling pathways.

• Journal of Microbiology and Biotechnology
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• v.10 no.5
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• pp.670-676
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• 2000

Several clones obtained from Serratia marcescens stimulated E. coli TP2139 (${\Delta}lac, \;{\Delta} crp$) cells to use maltose as a carbon source. The crp gene clone, pCKB12, was confirmed to stimulate the $\beta$-galactosidase activity, by Southern hybridization [31]. The nucleotide sequence of the crp region consisting of 1,979 bp was determined. The sequencing of the fragment led to the identification of two open reading frames: One of these, the crp gene, encoded 210 amino acid and the other encoded a truncated protein. The S. marcescens and E. coli crp genes showed a higher degree of divergence in their nucleotide sequence with 120 changes, however, the corresponding amino acid sequences showed only two amino acid differences. Yet, an analysis of the amino acid divergence revealed that the catabolite gene activator protein, the crp gene product, was the most conserved protein observed so far. Using a crp-lac protein fusion, it was demonstrated that S. marcescens CRP could repress its own expression, probably via a mechanism similar to that previously described for the E. coli crp gene.

• KCID journal
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• v.18 no.1
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• pp.33-43
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• 2011

Jundae reservoir has basin area of 234ha, average depth of 3.77m and total storage of $619{\times}10^3m^3$, and is located in Dangin-gun, Chungcheongnam-do. The water quality of Jundae reservoir exceeded the IV grade of water quality standard as available for irrigation water in COD, TN, TP, Chl-a. COD and Chl-a were higher in spring season, because the algal bloom by phytoplankton increased. And the algal blooms in October by inflow non-point pollution during summer rainy season. The most dominant zooplankton was rotifers during study period at all stations. Dominant species were Keratella cochlearis, Polyarthra spp., and Trichocerca spp. We successfully established 2 isolated clone cultures as predator. One is Rotifer, Euchlanis sp. and another is cladocerans, Bosmina sp. To test the removal rate of 2 cultures against Microcystis aeruginosa, we inoculated Euchlanis sp. and Bosmina sp. separately when the abundance reached at $1.0{\times}10^6$cells/ml. Euchlanis sp. removed M. aeruginosa around 98.9% and Bosmina sp. removed it around 98.4%. They are useful grazers for controling algae blooms, Euchlanis sp. and Bosmina sp. feeding on M. aeruginosa highly.

• Korean Journal of Agricultural Science
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• v.46 no.3
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• pp.471-488
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• 2019

Random genes were screened in two transforming ways to investigate the new genes of a ladybug using the Harmonia axyridis cDNA library stock cell cloned in the LITMUS 28i vector in a previous study. Phenotypic variation was observed after injection of the synthesized double-stranded RNA through the in vitro transcription process. The cDNA library of H. axyridis was transformed into E. coli $DH5{\alpha}$ and 10B competent cells by heat shock. Analysis of the nucleotide sequences of the 42 clones with the insert DNAs revealed that 21 clones were homologous with the genes of insects, and only one clone had a gene from H. axyridis. Thirteen of the 21 insect genes were homologous with genes from coleopteran insects. Fourteen genes were selected, which were identified by the gene screening results, and were synthesized as double-stranded RNA through in vitro transcription. One microgram of the synthesized double-stranded RNA between segments T1 and T2 were injected using a syringe into each anesthetized fourth larvae which were under 2 days old. As a result, a phenotypic variation appeared in the larva injected with the two genes. While the eggs of H. axyridis injected with distilled water hatched out three days after oviposition, the eggs of H. axyridis injected with dsHma 06 did not hatch but become shrivel a week after oviposition. Most of the H. axyridis injected with dsHma 08 died and were unable to complete the pupation or eclosion during ecdysis.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.04a
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• pp.98-98
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• 2019

This study was expand the use of Citrus madurensis (Citrofortunella microcarpa), which is a good source of skin whitening and antioxidants activation cosmetics. The goal was to provide cosmeceutical activity data about the extraction yield and total phenol of this calamondin orange by optimizing the focused high ultrasound(INEFU) and ultrasound extraction(UE) conditions. Under optimal extraction conditions, which consisted of 1800W for 45min and 500W for 45 min, 21.55 and 13.27 % (w/w) of the highest extractions yield and polyphenol contents were obtained. For the skin whitening activity, tyrosinase inhibitory activity was observed at 69.24 % in the INEFU extracts, which was ca. 18 % higher than that of the UE extracts (58.82 %). To reduce melanin production in Clone M-3 cells, 86.9 % melanin production was observed following the addition of control, WE(water extraction) relative to the control without a sample, and 81.2 and 78.9 % were found in the UE and INEFU conditions. The highest total phenol secretion was conclusively obtained under the optimal conditions and resulted in a significant improvement of the cosmetic activities of Citrus madurensis(Citrofortunella microcarpa) extracts.

• Journal of Microbiology and Biotechnology
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• v.32 no.5
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• pp.657-662
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• 2022

Glycosyltransferase (GT)-specific degenerate PCR screening followed by in silico sequence analyses of the target clone was used to isolate a member of family1 GT-encoding genes from the established fosmid libraries of soil actinomycetes Micromonospora echinospora ATCC 27932. A recombinant MeUGT1 was heterologously expressed as a His-tagged protein in E. coli, and its enzymatic reaction with semi-synthetic phenoxodiol isoflavene (as a glycosyl acceptor) and uridine diphosphate-glucose (as a glycosyl donor) created two different glycol-attached products, thus revealing that MeUGT1 functions as an isoflavonoid glycosyltransferase with regional flexibility. Chromatographic separation of product glycosides followed by the instrumental analyses, clearly confirmed these previously unprecedented glycosides as phenoxodiol-4'-α-O-glucoside and phenoxodiol-7-α-O-glucoside, respectively. The antioxidant activities of the above glycosides are almost the same as that of parental phenoxodiol, whereas their anti-proliferative activities are all superior to that of cisplatin (the most common platinum chemotherapy drug) against two human carcinoma cells, ovarian SKOV-3 and prostate DU-145. In addition, they are more water-soluble than their parental aglycone, as well as remaining intractable to the simulated in vitro digestion test, hence demonstrating the pharmacological potential for the enhanced bio-accessibility of phenoxodiol glycosides. This is the first report on the microbial enzymatic biosynthesis of phenoxodiol glucosides.