• Korean Journal of Fisheries and Aquatic Sciences
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• v.50 no.3
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• pp.302-310
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• 2017

Interferon regulatory factor 8 (IRF8) is essential for the development of B and T cells, as well as for the activity of dendritic cells and macrophages. We performed molecular characterization of IRF8 from rock fish, Sebastes schlegelii (Ss), and investigated the spatial and temporal profile of mRNA expression after challenge with lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C), or Streptococcus iniae. The full-length cDNA sequence of SsIRF8 was 1,657 bp, containing an ORF of 1,266 bp. The gene had a predicted molecular mass of 47.7 kDa and an isoelectric point of 5.99. The amino acid sequence coded by this gene showed the highest degree of identity (90.8%) and similarity (96.2%) with IRF8 from Oplegnathus fasciatus. The SsIRF8 mRNA was expressed ubiquitously, at varying levels, with the highest level of expression observed in the spleen. To confirm the role of SsIRF8 in mediating the immune response, we measured SsIRF8 mRNA expression in the splenic tissue at different time points after injection with LPS, poly I:C, or S. iniae. The qRT-PCR results showed that SsIRF8 mRNA expression in the poly I:C-injected group was highly upregulated 6 hr after exposure (P<0.05). Expression of SsIRF8 mRNA in the S. iniae-injected group peaked at 24 hr. These results suggest that SsIRF8 might be important in regulating the strength of the rockfish immune response to immunostimulatory agents.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.48 no.3
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• pp.354-361
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• 2015

Mitochondrial heat shock protein 75 (mtHSP75) is a member of the HSP90 family and plays essential roles in refolding proteins of the mitochondrial matrix. Mitochondria provide energy in the form of ATP and generate reactive oxygen species (ROS). Heat shock proteins (HSPs) are activated in response to stress, and protect cells. In this study, we characterized the mtHSP75 of the big-belly seahorse Hippocampus abdominalis. The protein (BsmtHSP75) is encoded by an open reading frame (ORF) of 2,157 nucleotides, has 719 amino acids (aa), and is of molecular mass 82 kDa. BsmtHSP75 has two functional domains, a histidine kinase-like ATPase (HATPase_c) domain (123-276 aa) and an HSP90 family domain (302-718 aa). BsmtHSP75 was expressed in all tested tissues of healthy seahorses. The ovary contained the highest transcription level, followed (in order) by the blood, brain, and muscle. Pouch tissue showed the lowest expression level. The expression of BsmtHSP75 was significantly (P<0.05) up-regulated on viral or bacterial challenge, suggesting that BsmtHSP75 plays a role in the immune defense against bacterial and viral pathogens.

• Journal of Microbiology and Biotechnology
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• v.24 no.7
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• pp.925-935
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• 2014

A cold-adapted carbohydrate esterase, CEST, belonging to the carbohydrate esterase family 6, was cloned from Microbulbifer thermotolerans DAU221. CEST was composed of 307 amino acids with the first 22 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the mature enzyme were 31,244 Da and pH 5.89, respectively. The catalytic triad consisted of residues Ser37, Glu192, and His281 in the conserved regions: GQSNMXG, QGEX(D/N), and DXXH. The three-dimensional structure of CEST revealed that CEST belongs to the ${\alpha}/{\beta}$-class of protein consisted of a central six-stranded ${\beta}$-sheet flanked by eight ${\alpha}$-helices. The recombinant CEST was purified by His-tag affinity chromatography and the characterization showed its optimal temperature and pH were $15^{\circ}C$ and 8.0, respectively. Specifically, CEST maintained up to 70% of its enzyme activity when preincubated at $50^{\circ}C$ or $60^{\circ}C$ for 6 h, and 89% of its enzyme activity when preincubated at $70^{\circ}C$ for 1 h. The results suggest CEST belongs to group 3 of the cold-adapted enzymes. The enzyme activity was increased by $Na^+$ and $Mg^{2+}$ ions but was strongly inhibited by $Cu^+$ and $Hg^{2+}$ ions, at all ion concentrations. Using p-nitrophenyl acetate as a substrate, the enzyme had a $K_m$ of 0.278 mM and a $k_{cat}$ of $1.9s^{-1}$. Site-directed mutagenesis indicated that the catalytic triad (Ser37, Glu192, and His281) and Asp278 were essential for the enzyme activity.

• Mass Spectrometry Letters
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• v.5 no.3
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• pp.63-69
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• 2014

In this study, we present a new isotope-coded carbamidomethylation (iCCM)-based quantitative proteomics, as a complementary strategy for conventional isotope labeling strategies, with providing the simplicity, ease of use, and robustness. In iCCM-based quantification, two proteome samples can be separately isotope-labeled by means of covalently reaction of all cysteinyl residues in proteins with iodoacetamide (IAA) and its isotope (IAA-$^{13}C_2$, $D_2$), denoted as CM and iCCM, respectively, leading to a mass shift of all cysteinyl residues to be + 4 Da. To evaluate iCCM-based isotope labeling in proteomic quantification, 6 protein standards (i.e., bovine serum albumin, serotransferrin, lysozyme, beta-lactoglobulin, beta-galactosidase, and alpha-lactalbumin) isotopically labeled with IAA and its isotope, mixed equally, and followed by proteolytic digestion. The resulting CM-/iCCM-labeled peptide mixtures were analyzed using a nLC-ESI-FT orbitrap-MS/MS. From our experimental results, we found that the efficiency of iCCM-based quantification is more superior to that of mTRAQ, as a conventional nonisobaric labeling method, in which both of a number of identified peptides from 6 protein standards and the less quantitative variations in the relative abundance ratios of heavy-/light-labeled corresponding peptide pairs. Finally, we applied the developed iCCM-based quantitative method to lung cancer serum proteome in order to evaluate the potential in biomarker discovery study.

• International Journal of Naval Architecture and Ocean Engineering
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• v.5 no.4
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• pp.580-597
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• 2013

Three materials SUS304, 9% Ni steel and Al 5083-O alloy, which are considered possible candidate for International Maritime Organization (IMO) type B Cargo Containment System, were studied. Monotonic tensile, fatigue, fatigue crack growth rate and Crack Tip Opening Displacement tests were carried out at room, intermediate low ($-100^{\circ}C$) and cryogenic ($-163^{\circ}C$) temperatures. The initial yield and tensile strengths of all materials tended to increase with decreasing temperature, whereas the change in elastic modulus was not as remarkable. The largest and smallest improvement ratio of the initial yield strengths due to a temperature reduction were observed in the SUS304 and Al 5083-O alloy, respectively. The fatigue strengths of the three materials increased with decreasing temperature. The largest increase in fatigue strength was observed in the Al 5083-O alloy, whereas the 9% Ni steel sample showed the smallest increase. In the fatigue crack growth rate test, SUS304 and Al 5083-O alloy showed a decrease in the crack propagation rate, due to decrease in temperature, but no visible improvement in da/dN was observed in the case of 9% Ni steel. In the Crack Tip Opening Displacement (CTOD) test, CTOD values were converted to critical crack length for the comparison with different thickness specimens. The critical crack length tended to decrease in the case of SUS304 and increase for the Al 5083-O alloy with decreasing temperature. In case of 9% Ni steel, change of critical crack length was not observed due to temperature decrease. In addition, the changing material properties according to the temperature of the LNG tank were analyzed according to the international code for the construction and equipment of ships carrying liquefied gases in bulk (IGC code) and the rules of classifications.

• BMB Reports
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• v.37 no.4
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• pp.408-415
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• 2004

The expression of the Paenibacillus sp. A11 cyclodextrinase (CDase) gene using the pUC 18 vector in Escherichia coli JM 109 resulted in the formation of an insoluble CDase protein in the cell debris in addition to a soluble CDase protein in the cytoplasm. Unlike the expression in Paenibacillus sp. A11, CDase was primarily observed in cytoplasm. However, by adding 0.5 M sorbitol as an osmolyte, the formation of insoluble CDase was prevented while a three-fold increase in cytoplasmic CDase activity was achieved after a 24 h-induction. The recombinant CDase protein was purified to approximately 14-fold with a 31% recovery to a specific activity of 141 units/mg protein by 40-60% ammonium sulfate precipitation, DEAE-Toyopearl 650 M, and Phenyl Sepharose CL-4B chromatography. It was homogeneous by non-denaturing and SDS-PAGE. The enzyme was a single polypeptide with a molecular weight of 80 kDa, as determined by gel filtration and SDS-PAGE. It showed the highest activity at pH 7.0 and $40^{\circ}C$. The catalytic efficiency ($k_{cat}/K_m$) values for $\alpha$-, $\beta$-, and $\gamma$-CD were $3.0{\times}10^5$, $8.8{\times}10^5$, and $5.5{\times}10^5\;M^{-1}\;min^{-1}$, respectively. The enzyme hydrolyzed CDs and linear maltooligosaccharides to yield maltose and glucose with less amounts of maltotriose and maltotetraose. The rates of hydrolysis for polysaccharides, soluble starch, and pullulan were very low. The cloned CDase was strongly inactivated by N-bromosuccinimide and diethylpyrocarbonate, but activated by dithiothreitol. A comparison of the biochemical properties of the CDases from Paenibacillus sp. A11 and E. coli transformant (pJK 555) indicates that they were almost identical.

• Animal Bioscience
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• v.35 no.12
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• pp.1921-1928
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• 2022

Objective: This research aimed to evaluate the effects of age on growth, tibia development, and intestinal calcium (Ca) and phosphorus (P) transporter gene expressions in broiler chickens. Methods: A total of 224 male Arbor Acres broilers were fed with nutrient-adequate diets and reared in eight cages (28 broilers per cage). Eight broilers (one broiler per cage) were selected and killed at 5, 10, 15, 20, 25, 30, 35, and 40 days of age, respectively. Results: Body weight continuously increased with age of broiler chickens from 5 to 40 days. The bone weight, ash weight, diameter, and length of the tibia also increased with broiler age. By contrast, the tibia ash, Ca, and P percentages quadratically changed with age (p<0.001), and the highest values of mineral contents were observed at 20, 25, and 25 days of age, respectively. The mRNA abundances of calcium-binding protein 28-kDa (CaBP-D28k), sodium-calcium exchanger 1 (NCX1), and plasma membrane ATPase 1b (PMCA1b) increased from 5 to 25 days and then decreased up to 40 days. Similar results were noted in the mRNA abundances of IIb sodium-phosphate cotransporter (NaPi-IIb), inorganic phosphate transporter 1 (PiT-1), inorganic phosphate transporter 2 (PiT-2), nuclear vitamin D receptor (nVDR), and membrane vitamin D receptor (mVDR). The mRNA abundances of Ca and P transporters and VDRs were the highest at 25 days of age. Conclusion: These data indicate that age quadratically affects intestinal Ca and P transporter gene expression and mineral absorption capacity in broiler chickens.

• Proceedings of the Korea Committee for Ocean Resources and Engineering Conference
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• 2002.05a
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• pp.155-160
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• 2002

Metal matrix composites had generated a lot of interest in recent times because of significant in specific properties. It was also highlighted as the materials of frontier industry because strength, heat-resistant, corrosion-resistant, wear-resistant were superiored. In this study the strength properties of $Al_{18}B_4O_{33}/AC4CH$ were represented mixing the binder of $Al_2O_3$ and $TiO_2$. It was also fabricated by squeeze casting. $Al_{18}B_4O_{33}/AC4CH$ was fabricated at the melt temperature of $760^{\circ}C$ the perform temperature of $700^{\circ}C$ and mold temperature of $200^{\circ}C$ under the pressure of 83.4MPa and observed SEM. Fatigue crack growth rate tests on compact tension specimen(half-size) of thickness 12.5mm were conducted by using sinusoidal waveform. Compact tension specimens(half-size) were used and fatigue crack growth rate da/dN and stress intensity factor range ${\Delta}K$ were analyzed concerning to the R value of 0.1 and 0.05. In order to find out the value of ${\Delta}K$, load amplitude constant method was applied by the standard fatigue testing method describes in ASTM E647-95a. As the results of this study, Fatigue crack growth rate increased with in creasing the load ratio, Consequently, At equivalent stress intensity factors, the fatigue crack growth rates in MMC were faster than those of AC4CH alloy. then the fatigue life and the fatigue crack growth rate was investigated using scanning election microscopy(SEM)

• Toxicological Research
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• v.17
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• pp.97-104
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• 2001

Three types of proteases (MEF-1, MEF-2 and MEF-3) were purified from the egg cases of Ten-odera sinensis using ammonium sulfate fractionation, gel filtration on Bio-Gel P-60 and affinity chromatography on DEAE Affi-Gel blue gel. The proteases were assessed homogeneous by SDS-polyacrylamide gel electrophoresis and have molecular weight of 31,500, 32,900 and 35,600 Da, respectively. The N-terminal regions of the primary structure were compared and they were found to be different each other. MEFs readily digested the $A\alpha$ - and B$\beta$-chains of fibrinogen and more slowly the ${\gamma}$-chain. The action of the enzymes resulted in extensive hydrolysis of fibrinogen and fibrin, releasing a variety of fibrinopeptides. MEF-1 was inactivated by Cu$^{2+}$ and Zn$^{2+}$ and inhibited by PMSF and chymostatin. MEF-2 was inhibited by PMSF, TLCK. soybean trypsin inhibitor. MEF-3 was only inhibited by PMSF and chymostatin. Antiplasmin was not sensitive to MEF-1 but antithrombin III inhibited the enzymatic activity qf MEF-1. MEF-2 specifically bound to anti plasmin Among the chromogenic protease substrates, the most sensitive one to the hydrolysis of MEFs was benzoyl-Phe-Val-Arg-p-nitroanilide with maximal activity at pH 7.0 and 3$0^{\circ}C$. MEF-1 preferentially cleaved the oxidized B-chain of insulin between Leu15 and Tyr16. In contrast, MEF-2 specifically cleaved the peptide bond between Arg23 and Gly24. D-dimer concentrations increased on incubation of cross-linked fibrin with MEF-1, indicating the enzyme has a strong fibrinolytic activity.ity.

• Journal of Applied Biological Chemistry
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• v.56 no.2
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• pp.89-93
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• 2013

Gamma-aminobutyric acid (GABA) aminotransferase (gabT) and succinic semialdehyde dehydrogenase (gabD) genes from Pseudomonas fluorescens KCCM 12537 were cloned into a single pETDuet-1 vector and co-expressed in Escherichia coli BL21(DE3) simultaneously. The mixture of both enzymes, called GABase, is the key enzyme for the enzymatic analysis of GABA. The molecular mass of the GABA aminotransferase and succinic semialdehyde dehydrogenase were determined to be 52.8 and 46.7 kDa following computations performed with the pI/Mw program, respectively. The GABase activity between pH 6.0 and 9.0 for 24 h at $4^{\circ}C$ remained over 75%, but under pH 6.0 decreased rapidly. The GABase activity between 25 and $35^{\circ}C$ by the treatment at pH 8.6 for 30 min remained over 80%, but over $35^{\circ}C$ decreased rapidly. When the activity against GABA was defined as 100%, the purified GABase activity against 5-aminovaleric acid having a similar structure to GABA showed 47.7% and GABase activity against ${\beta}$-alanine, ${\varepsilon}$-amino-n-caproic acid, $_L$-ornithine, $_L$-lysine, and $_L$-aspartic acid showed between 0.3 to 2.3%. The GABA content was analyzed with this co-expressed GABase, compared with the other GABase which was available commercially. As a result, the content of GABA extracted from brown rice, dark brown rice, and black rice were $26.4{\pm}3.5$, $40.5{\pm}4.7$ and $94.7{\pm}9.3{\mu}g/g$, which were similar data of other GABase in the error ranges.