• Journal of Life Science
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• v.16 no.7 s.80
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• pp.1133-1140
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• 2006

Human HtrA1 (High temperature requirement protein A1) is a homologue of the E. coli periplasmic serine protease HtrA. A recent study has demonstrated that HtrA1 is a serine protease involved in processing of insulin like growth factor binding protein (ICFBP), indicating that it serves as an important regulator of IGF activity. Additionally, several lines of evidence suggest a striking correlation between proteolytic activity of HtrA1 serine protease and the pathogenesis of several diseases; however, physiological roles of HtrA1 remain to be elucidated. We used the pGEX bacterial expression system to develop a simple and rapid method for purifying HtrA1, and the recombinant HtrA1 protein was utilized to investigate the optimal conditions in executing its proteolytic activity. The proteolytically active HtrA1 was purified to approximately 85% purity, although the yield of the recombinant HtrA1 protein was slightly low $460{\mu}g$ for 1 liter E. coli culture). Using in vitro endoproteolytic cleavage assay, we identified that the HtrA1 serine protease activity was dependent on the enzyme concentration and the incubation time and that the best reaction temperature was $42^{\circ}C$ instead of $37^{\circ}C$. We arbitrary defined one unit of proteolytic activity of the HtrA1 serine protease as 200nM of HtrA1 that cleaves half of $5{\mu}M\;of\;{\beta}-casein$ during 3 hr incubation at $37^{\circ}C$. Our study provides a method for generating useful reagents to investigate the molecular mechanisms by which HtrA1 serine protease activity contributes in regulating its physiological function and to identify natural substrates of HtrA1.

• Journal of Life Science
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• v.16 no.5
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• pp.716-722
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• 2006

Superoxide dismutase (SOD) is physiologically important in regulating cellular homeostasis and apoptotic cell death, and its mutations are the cause of familial amyotrophic lateral sclerosis (FALS). Mitochondrial serine protease HtrA2 has a pro-apoptotic function and has known to be associated with neurodegenerative disorders. To investigate the relationship between genes associated with apoptotic cell death, such as HtrA2 and SOD1, we utilized the pGEX expression system to develop a simple and rapid method for purifying wild-type and ALS-associated mutant SOD1 proteins in a suitable form for biochemical studies. We purified SOD1 and SOD1 (G93A) proteins to approximately 90% purity with relatively high yields (3 mg per liter of culture). Consistent with the result in mammalian cells, SOD1 (G93A) was more insoluble than wild-type SOD1 in E. coli, indicating that research on the aggregate formation of SOD1 may be possible using this pGEX expression system in E. coli. We investigated the HtrA2 serine protease activity on SOD1 to assess the relationship between two proteins. Not only wild-type SOD1 but also ALS-associated mutant SOD1 (G93A) were cleaved by HtrA2, resulting in the production of the 19 kDa and 21 kDa fragments that were specific for anti-SOD1 antibody. Using protein gel electrophoresis and immunoblot assay, we compared the relative molecular masses of thrombin-cleaved GST-SOD1 and HtrA2-cleaved SOD1 fragments and can predict that the HtrA2-cleavage sites within SOD1 are the peptide bonds between leucine 9-lysine 10 (L9-K10) and glutamine 23-lysine 24 (Q23-K24). Our study indicates that SOD1 is one of the substrate for HtrA2, suggesting that both HtrA2 and SOD1 may be important for modulating the HtrA2-SOD1-mediated apopotic cell death that is associated with the pathogenesis of neurodegenerative disorder.

• Biomedical Science Letters
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• v.21 no.4
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• pp.181-187
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• 2015

Osteoporosis is one of the diseases caused by accumulation of effects from complex interactions between genetic and environmental factors. Aging is the major cause for osteoporosis, which normally increases skeletal fragility and bone fracture especially among the elder. "Omics" refers to a specialized research field dealing with high-throughput biological data, such as genomics, transcriptomics, proteomics or metabolomics. Integration of data from multi-omics has been approved to be a powerful strategy to colligate biological phenomenon with multiple aspects. Actually, integrative analyses of "omics" datasets were used to present pathogenesis of specific diseases or casual biomarkers including susceptible genes. In this study, we evaluated the proposed relationship of novel susceptible genes (TREML2, HTR1E, and GLO1) with osteoporosis, which genes were obtained using multi-omics integration analyses. To this end, SNPs of the susceptible genes in the Korean female cohort were analyzed. As a result, one SNP of HTR1E and five SNPs of TREML2 were identified to associate with osteoporosis. The highest significant SNP was $rs6938076^*$ of TREML2 (OR=0.63, CI: 0.45~0.89, recessive P=0.009). Consequently, the susceptible genes identified through the multi-omics analyses were confirmed to have association with osteoporosis. Therefore, multi-omics analysis might be a powerful tool to find new genes associated with a disease. We further identified that TREML2 has more associated with osteoporosis in females than did HTR1E.

• Bulletin of the Korean Chemical Society
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• v.32 no.5
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• pp.1692-1696
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• 2011

The reactions of 1H-1,2,4-triazole (Htr) with $MX_2$ ($ZnCl_2$ for 1; $CdBr_2$ for 2) resulted in two coordination polymers, [Zn(tr)Cl]$_n$ (1) and $[Cd(Htr)_2Br_2]_n$ (2). The structural analyses indicate that 1 and 2 feature a 2D layer and 1D triple chain, respectively. In 1, neighouring Zn atoms are connected by ${\mu}_3-1$ ${\kappa}N$: 2 ${\kappa}N$: $4{\kappa}N-tr^-$ anionic ligand into 6- and 16-membered rings, further grow into a 2D sheet. Cd atoms in 2 are bonded by two ${\mu}_2-Br^-$ bridges and neutral ${\mu}_2$-1 ${\kappa}N$: 2 ${\kappa}N$-Htr to form a 1D triple chain. The fluorescent characterizations of 1, 2 and the free Htr ligand feature simlilar emission peakes at 444, 446 and 423 nm respectively, which can be assigned to intra-ligand ${\pi}-{\pi}^*$ transition of (H)tr. The energy gaps of 5.90 eV for 1, 5.16 eV for 2, and 5.93 eV for Htr suggest that the compounds behave as insulators.

• Molecules and Cells
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• v.20 no.1
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• pp.83-89
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• 2005

HtrA2/Omi is a mammalian mitochondrial serine protease homologous to the E. coli HtrA/DegP gene products. Recently, HtrA2/Omi was found to have a dual role in mammalian cells, acting as an apoptosis-inducing protein and being involved in maintenance of mitochondrial homeostasis. By screening a human brain cDNA library with $A{\beta}$ peptide as bait in a yeast two-hybrid system, we identified HtrA2/Omi as a binding partner of $A{\beta}$ peptide. The interaction between $A{\beta}$ peptide and HtrA2/Omi was confirmed by an immunoblot binding assay. The possible involvement of HtrA2/Omi in $A{\beta}$ peptide metabolism was investigated. In vitro peptide cleavage assays showed that HtrA2/Omi did not directly promote the production of $A{\beta}$ peptide at the ${\beta}/{\gamma}$-secretase level, or the degradation of $A{\beta}$ peptide. However, overexpression of HtrA2/Omi in K269 cells decreased the production of $A{\beta}40$ and $A{\beta}42$ by up to 30%. These results rule out the involvement of HtrA2/Omi in the etiology of Alzheimer's disease. However, the fact that overexpression of HtrA2/Omi reduces the generation of $A{\beta}40$ and $A{\beta}42$ suggests that it may play some positive role in mammalian cells.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.135.2-136
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• 2003

To characterize plasmids in Xanthomonu axonopodis pv. glycines, we isolated plasmids pAG1 from the strain AG1 and pXAG81 and PXAG82 from the strain Bra, respectively, and sequenced three plasmids. The size of plasmids, pAG1, pXAG81, and pXAG82 was 15,149-base pairs (bp), 26,727-bp, and 1,496-bp, respectively Fifteen and twenty six possible open reading frames (ORFs) were present in pAG1 and pXAG81, respectively. Only one ORF homologous to a rep gene of Xylella fastidiosa was present in pXAG82. pAG1 contained genes homologous to avrBs3, tnpA, tnpR, repA, htrA, three parA genes, M.XmaI, R.XmaI, and six hypothetical proteins. pXAG81 contained genes homologous to avrBs3, tnpA, tnpR, repA, htrA, two parA genes, pemI, pemK, mobA, mobB, mobC, mobD, mobE, trwB, traF, traH, ISxac2, and eleven hypothetical proteins. Based on DNA sequence analysis, we presume that pXAG81 is a conjugal plasmid. Interestingly, we found 0.5-kb truncated avirulence gene similar to aurXacE3 on the right border of avrBs3 homolgs of pAG1 and pXAG81. Two hundred twenty five isolates were analyzed to find aurBS3 or tra gene homologs by Southern hybridization. The numbers of avrBs3 homolog varied from 3 in AG1 to 8 in AG166. Two hundred seventeen isolates appeared to can conjugative plasmids (pXAG81 type), and thirty eight isolates appeared to carry non-conjugative plamids (pAGl type). This indicated that aurBs3 gene homologs might be spread by conjugation in X. axonopodis pv. glycines.

• Journal of Animal Science and Technology
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• v.64 no.5
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• pp.922-936
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• 2022

5-Hydroxytryptamine (5-HT), a monoamine, as a local regulator in the mammary gland is a chemical signal produced by the mammary epithelium cell. In cows, studies have shown that 5-HT is associated with epithelial cell apoptosis during the degenerative phase of the mammary gland. However, studies in other tissues have shown that 5-HT can effectively promote cell viability. Whether 5-HT could have an effect on mammary cell viability in dairy cows is still unknown. The purpose of this study was to determine: (1) effect of 5-HT on the viability of bovine mammary epithelial cells and its related signaling pathways, (2) interaction between prolactin (PRL) and 5-HT on the cell viability. The bovine mammary alveolar cell-T (MAC-T) were cultured with different concentrations of 5-HT for 12, 24, 48 or 72 hours, and then were assayed using cell counting kit-8, polymerase chain reaction (PCR) and immunobloting. The results suggested that 20 μM 5-HT treatment for 12 or 24 h promote cell viability, which was mainly induced by the activation of 5-HT receptor (5-HTR) 1B and 4, because the increase caused by 5-HT vanished when 5-HTR 1B and 4 was blocked by SB224289 and SB204070. And protein expression of mammalian target of rapamycin (mTOR), eukaryotic translation elongation factor 2 (eEF2), janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) were decreased after blocking 5-HT 1B and 4 receptors. When MAC-T cells were treated with 5-HT and PRL simultaneously for 24 h, both the cell viability and the level of mTOR protein were significantly higher than that cultured with 5-HT or PRL alone. In conclusion, our study suggested that 5-HT promotes the viability of MAC-T cells by 5-HTR 1B and/or 4. Furthermore, there is a reciprocal relationship between PRL and 5-HT.

• Nuclear Engineering and Technology
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• v.45 no.2
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• pp.211-218
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• 2013

Large quantities of irradiated graphite waste from graphite-moderated nuclear reactors exist and are expected to increase in the case of High Temperature Reactor (HTR) deployment [1,2]. This situation indicates the need for a graphite waste management strategy. Of greatest concern for long-term disposal of irradiated graphite is carbon-14 ($^{14}C$), with a half-life of 5730 years. Fachinger et al. [2] have demonstrated that thermal treatment of irradiated graphite removes a significant fraction of the $^{14}C$, which tends to be concentrated on the graphite surface. During thermal treatment, graphite surface carbon atoms interact with naturally adsorbed oxygen complexes to create $CO_x$ gases, i.e. "gasify" graphite. The effectiveness of this process is highly dependent on the availability of adsorbed oxygen compounds. The quantity and form of adsorbed oxygen complexes in pre- and post-irradiated graphite were studied using Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and Xray Photoelectron Spectroscopy (XPS) in an effort to better understand the gasification process and to apply that understanding to process optimization. Adsorbed oxygen fragments were detected on both irradiated and unirradiated graphite; however, carbon-oxygen bonds were identified only on the irradiated material. This difference is likely due to a large number of carbon active sites associated with the higher lattice disorder resulting from irradiation. Results of XPS analysis also indicated the potential bonding structures of the oxygen fragments removed during surface impingement. Ester- and carboxyl-like structures were predominant among the identified oxygen-containing fragments. The indicated structures are consistent with those characterized by Fanning and Vannice [3] and later incorporated into an oxidation kinetics model by El-Genk and Tournier [4]. Based on the predicted desorption mechanisms of carbon oxides from the identified compounds, it is expected that a majority of the graphite should gasify as carbon monoxide (CO) rather than carbon dioxide ($CO_2$). Therefore, to optimize the efficiency of thermal treatment the graphite should be heated to temperatures above the surface decomposition temperature increasing the evolution of CO [4].

• The Journal of the Korean Society for Microbiology
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• v.35 no.2
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• pp.149-157
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• 2000

Mycobacterium tuberculosis is capable of growing and survival within macrophage. The purpose of this study was to identify the genes regulated by infection of mycobacteria in human monocytic THP-1 cells. We used the differential display reverse transcriptase polymerase chain reaction (DD RT-PCR) and nothern blot analysis to confirm the differentially expressed genes from THP-1 cells infected with live Mycobacterium tuberculosis H37Rv, heat-killed Mycobacterium tuberculosis H37Rv and live Mycobacterium bovis BCG. Among many up or down-regulated clones, 27 clones were sequenced and compared with known genes on GenBank. Thirteen of over-expressed clones from THP-1 cells infected with live Mycobacterium tuberculosis H37Rv were identical to human prothymosin alpha, eight were novel clones and six clones showed homology with Human ferritin H chain, Esherichia coli bgl, Mouse RNA-dependent EIF-2 alpha kinase, E. coli htrL, Hyaluronan receptor and T cell receptor. Our result suggests that Mycobacterium tuberculosis might regulate prothymosin alpha gene transcription in monocytic THP-1 cell.

• Nuclear Engineering and Technology
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• v.39 no.5
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• pp.603-616
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• 2007

Roy Huddle, having invented the coated particle in Harwell 1957, stated in the early 1970s that we know now everything about particles and coatings and should be going over to deal with other problems. This was on the occasion of the Dragon fuel performance information meeting London 1973: How wrong a genius be! It took until 1978 that really good particles were made in Germany, then during the Japanese HTTR production in the 1990s and finally the Chinese 2000-2001 campaign for HTR-10. Here, we present a review of history and present status. Today, good fuel is measured by different standards from the seventies: where $9*10^{-4}$ initial free heavy metal fraction was typical for early AVR carbide fuel and $3*10^{-4}$ initial free heavy metal fraction was acceptable for oxide fuel in THTR, we insist on values more than an order of magnitude below this value today. Half a percent of particle failure at the end-of-irradiation, another ancient standard, is not even acceptable today, even for the most severe accidents. While legislation and licensing has not changed, one of the reasons we insist on these improvements is the preference for passive systems rather than active controls of earlier times. After renewed HTGR interest, we are reporting about the start of new or reactivated coated particle work in several parts of the world, considering the aspects of designs/ traditional and new materials, manufacturing technologies/ quality control quality assurance, irradiation and accident performance, modeling and performance predictions, and fuel cycle aspects and spent fuel treatment. In very general terms, the coated particle should be strong, reliable, retentive, and affordable. These properties have to be quantified and will be eventually optimized for a specific application system. Results obtained so far indicate that the same particle can be used for steam cycle applications with $700-750^{\circ}C$ helium coolant gas exit, for gas turbine applications at $850-900^{\circ}C$ and for process heat/hydrogen generation applications with $950^{\circ}C$ outlet temperatures. There is a clear set of standards for modem high quality fuel in terms of low levels of heavy metal contamination, manufacture-induced particle defects during fuel body and fuel element making, irradiation/accident induced particle failures and limits on fission product release from intact particles. While gas-cooled reactor design is still open-ended with blocks for the prismatic and spherical fuel elements for the pebble-bed design, there is near worldwide agreement on high quality fuel: a $500{\mu}m$ diameter $UO_2$ kernel of 10% enrichment is surrounded by a $100{\mu}m$ thick sacrificial buffer layer to be followed by a dense inner pyrocarbon layer, a high quality silicon carbide layer of $35{\mu}m$ thickness and theoretical density and another outer pyrocarbon layer. Good performance has been demonstrated both under operational and under accident conditions, i.e. to 10% FIMA and maximum $1600^{\circ}C$ afterwards. And it is the wide-ranging demonstration experience that makes this particle superior. Recommendations are made for further work: 1. Generation of data for presently manufactured materials, e.g. SiC strength and strength distribution, PyC creep and shrinkage and many more material data sets. 2. Renewed start of irradiation and accident testing of modem coated particle fuel. 3. Analysis of existing and newly created data with a view to demonstrate satisfactory performance at burnups beyond 10% FIMA and complete fission product retention even in accidents that go beyond $1600^{\circ}C$ for a short period of time. This work should proceed at both national and international level.