• BMB Reports
• /
• v.36 no.3
• /
• pp.299-304
• /
• 2003

For deciphering the cyclic guanosine monophosphate (cGMP) signaling pathway, we employed chemical proteomics to identify the novel target molecules of cGMP. We used cGMP that was immobilized onto agarose beads with linkers directed at three different positions of cGMP. We performed a pull-down assay using the beads as baits on tissue lysates and identified 9 proteins by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry. Some of the identified proteins were previously known cGMP targets, including cGMP-dependent protein kinase and cGMP-stimulated phosphodiesterase. Surprisingly, some of the co-precipitated proteins were never formerly reported to associate with the cGMP signaling pathway. The competition binding assays showed that the interactions are not by nonspecific binding to either the linker or bead itself, but by specific binding to cGMP. Furthermore, we observed that the interactions are highly specific to cGMP against other nucleotides, such as cyclic adenosine monophosphate (cAMP) and 5'-GMP, which are structurally similar to cGMP. As one of the identified targets, MAPK1 was confirmed by immunoblotting with an anti-MAPK1 antibody. For further proof, we observed that the membrane-permeable cGMP (8-bromo cyclic GMP) stimulated mitogen-activated protein kinase 1 signaling in the treated cells. Our present study suggests that chemical proteomics can be a very useful and powerful technique for identifying the target proteins of small bioactive molecules.

• Development and Reproduction
• /
• v.9 no.1
• /
• pp.43-48
• /
• 2005

Bone marrow stromal cells, a constituent of the niche for hematopoietic stem cells in bone marrow, provide various factors involved in the fate decision of the hematopoietic stem and progenitor cells. Radiation, a widely used anti-cancer therapy, provokes side effects including the damage of the blood cells. Therefore, it is necessary to recover the blood cells shortly after radiation via promoting the differentiation of hematopoietic cells. In this study, we screened genes modulated by radiation in human bone marrow stromal cells in order to understand the mechanism involved in hematopoiesis after radiation. We performed differential display method by using polymerase chain reaction(PCR) and agarose gel electrophoresis. We found plasminogen activator inhibitor-1(PAI-1) was consistently induced by radiation. The significance of the PAI-1 gene modulation is to be determined.

• Microbiology and Biotechnology Letters
• /
• v.14 no.2
• /
• pp.155-160
• /
• 1986

$\alpha$-Amylase gene of B. amyloliquefaciens was cloned to E. coli-yeast shuttle vector YEp-13 and expressed in E. coli. Chromosomal DNA of B. amyloliquefaciens was partially digested with Sau3Al and YEp13 plasmid was cleaved with BamH1. The hybrid plasmid, pHA28, was constructed by shotgun method and transformed to E. coli C600 and HB101. The amount of $\alpha$-amylase produced by transformants of E. coli was about 20% to 30% of that produced by B. amyloli-quefaciens. About 65% of $\alpha$-amylase produced by transformant was secreted into periplasm and the others were located in cytoplasm. $\alpha$-Amylase production was maximal when transformants were cultivated for 15hr to 20hr. As the result of agarose gel electrophoresis, pHA28 plasmid was found to be various in its size. This result suggested that pHA28 plasmid was segregated.

• Microbiology and Biotechnology Letters
• /
• v.47 no.4
• /
• pp.662-666
• /
• 2019

In this study, the NABH558 gene expression system was constructed to efficiently produce neoagarobiose hydrolase (NABH) in Saccharomyces cerevisiae strain. The ADH1 and GAL10 promoters of the pAMFα-NABH and pGMFα-NABH plasmids were examined to determine the suitable promoter for the NABH558 gene expression, respectively. The effect of promoter and carbon sources on NABH558 gene expression was investigated by transforming each plasmid into the S. cerevisiae 2805 strain. The NABH activity in the 2805/pAMFα-NABH strain was 0.069 unit/ml/DCW in YPD medium, whereas that in the 2805/pGMFα-NABH strain was similar (0.02-0.027 unit/ml/DCW) irrespective of the medium composition. The higher NABH activity in the YPD medium was due to the increased NABH558 gene transcription. NABH produced in the recombinant strains could degrade agarose to galactose and AHG. This indicated that ADH1 promoter was a more optimal promoter for the expression of NABH558 gene than the GAL10 promoter. The NABH activity induced by the ADH1 promoter was about 3-fold higher than that induced by the GAL10 promoter.

• Korean Journal of Food Science and Technology
• /
• v.22 no.2
• /
• pp.134-139
• /
• 1990

A strain of Nuruk yeast No. IS (NY-15) which produced high activity of ${\beta}-galactosidase$ was isolated from Nuruk, and the crude enzyme was prepared by whey permeate culture of the microorganism. The crude enzyme was purified 40-fold with a 7.7% yield by acetone and ammonium sulfate fractionational precipitation, and chromatography on DEAE-cellulose, DEAE-Sephadex A-50 and Agarose-PAPT. Purified ${\beta}-galactosidase$ from Nuruk yeast showed two types of subunit patterns; a slow moving band and a fast moving deeply stained band, both anode·migrating at pH 7.5. The molecular weight of the former was estimated to be about 130,000 and that of the latter was 96,000 by SDS-polyacrylamide gel electrophoresis. The optimum pH of the enzyme activity was 7.5 and maximum activity appeared at $40^{\circ}C$.

• Journal of Biomedical Engineering Research
• /
• v.30 no.4
• /
• pp.333-340
• /
• 2009

MR compatible coaxial-slot antenna for microwave hyperthermia was developed while its structure and size of each part were determined by computer simulation using finite element method(FEM). Its local heating performance was evaluated using tissue-mimic phantom and swine muscles. 2% agarose gel mixed with 6mM/$\ell$ $MnCl_2$ as a biological tissue-mimic phantom was heated by the proposed antenna driven by a 2.45GHz microwave generator. The temperature changes of the phantom were monitored using multi-channel digital thermometer at the distance of 0mm, 5mm, 10mm and 20mm from the tip center of the antenna. Also muscle tissue of swine was heated for 2 and 5minutes with 50W and 30W of microwave generator powers, respectively, to evaluate the local heating performance of the antenna. MRI compatibility was also verified by acquiring MR images and MR temperature map. MR signals were acquired from the agarose gel phantom using $T2^*$ GRE sequence with 1.5T clinical MRI scanner(Signa Echospeed, GE, Milwaukee, WI, U.S.A.) at Pusan Paik Hospital and were transferred to PC in order to reconstruct MR images and temperature map using proton resonance frequency(PRF) method and laboratory-developed phase unwrapping algorithm. Authors found that it has no severe distortion due to the antenna inserted into the phantom. Finally, we can conclude that the suggested coaxial-slot antenna has an excellent local heating performance for both of tissue-mimic phantom and swine muscle, and it is compatible to 1.5T MRI scanner.

• Journal of Korean Ophthalmic Optics Society
• /
• v.5 no.1
• /
• pp.83-87
• /
• 2000

To investigate exhausted-medium-induced apoptosis in human corneal epithelial(HCE) cells, this study was performed DNA gel electrophoresis, M30 CytoDEATH staining and FAS-FAS ligand ELISA. SV-40 transfected cells were grown to confluency in culture for 7days. The supernatant was harvested and filtered with $0.22{\mu}m$ filter paper. Fresh HCE cells were exposed to the filtered exhausted medium for 1~2 days. Apoptotic cells were prepared for DNA extraction and run the agarose gel for DNA ladder pattern. M30 CytoDEATH was used a tool for easy and reliable determination of very early apoptosis in HCE cells. The control and exhausted medium were assayed for soluble FAS/FAS ligand protein by ELISA. HCE cells exposed to exhausted medium showed a typical DNA ladder pattern. Sporadic M30 CytoDEATH positive cells were detected among HCE cells exposed to exhausted medium. Soluble FAS/FAS ligand levels were not elevated in the exhausted medium compared to the fresh medium control. This study suggests that possible mechanism of exhausted medium induced apoptosis does not include the FAS-FAS ligand system.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.9
• /
• pp.1237-1244
• /
• 2012

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and $42.5^{\circ}C$, and the enzyme was stable under $40^{\circ}C$. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a ${\beta}$-agarase.

• KSBB Journal
• /
• v.14 no.6
• /
• pp.702-706
• /
• 1999

The genetic responses of aquaculturable seaweed Prophyra yezoensis tissue by acid shock have been compared using differential display technique. The tissue has been challenged in seawater containing 0.05% hydrogen chloride(pH 3.0) for 5 min, then rehabilitated in normal seawater for 10 min, 30 min, 60 min and 4 hrs, respectively. Total RNA was extracted by LiCl-guanidinium method. The cDNA was synthesized by reverse transcription with random hexamers and amplified by PCR with arbitrary primers. The genetic fragment disappeared by acid shock was selectively isolated from agarose gel and sequenced with a DNA auto sequencer. One of the acid-labile gene(605 bp) was identified as a dethiobiotin synthetase gene according to sequence alignment analysis by the NCBI BLAST search program.

• Korean journal of applied entomology
• /
• v.36 no.3
• /
• pp.264-269
• /
• 1997

Four strains of Bacillus thuringiensis were isolated froin the dead larvae of mulberry longicorn beetle (Apriong germari) and dung beetle (Aphodius apicalis). One nf four B. thuringiensis isolates turned out to be subspecies darinstadiensis but the remains were not identified using 33 B. thuringiensts flgellar ( H ) antibodies. Furthermore. bioassays of spore-parasporal inclusion protein mixture conducted against third instar larvae of A. gerrntrri or A. apicalis, second instar larvae of Bombyx mori, and third instar larvae of Cu1ex pipiens pullens showed that the isolates were non-toxic. To further confirm, four isolates were characterized and analysed by SDS-PAGE and agarose gel electrophoresis. The results revealed that parasporal protein and plasmid DNA patterns of four isolates are different from those of darmstadiensis and 20 known non-toxic B. thuringiensis strains, suggesting that the four isolates are novel non-toxic B. thuringiensis.