• Journal of the Korean Society of Food Science and Nutrition
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• v.33 no.10
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• pp.1668-1675
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• 2004

The effect of pH on surface hydrophobicity, sulfhydryl group, infrared spectrum, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) pattern and enthalpy was investigated in recovered protein from mackerel and frozen blackspotted croaker by alkaline processing. Hydrophobic residue in myofibrillar protein exposed to the surface of protein, and hydrophobic interaction were the highest around 6$0^{\circ}C$. The surface hydrophobicity was different between myofibrillar protein and myofibrillar protein including sarcoplasmic protein (recovered protein). The peak at 1636 c $m^{-l}$ was increased with pH, and the recovered protein was unfolded in alkali pH. Difference of surface and total sulfhydryl group at pH 7.0 and 10 was comparative high, and decrease of surface sulfhydryl group indicated formation of S-S bonds. Mackerel and frozen blackspotted croaker in alkaline pH showed bands of polymerized myosin heavy chain on SDS-PAGE pattern. The transition temperatures of recovered protein were 33.1, 44.3 and 65.5$^{\circ}C$. Gelation of recovered protein from alkali processing was estimated by increase of $\beta$-sheet structure by pH treatment, S-S bonds by oxidation of surface sulfhydryl group in heating, polymerization of myosin heavy chain in order.r.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1997.04a
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• pp.95-95
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• 1997

Among the various receptor molecules discovered so far the ${\beta}$2-adrenergic receptors have been regarded as excellent model systems for the so called 7 transmembrane helix receptor and have been the focus of extensive studies. For the analysis of receptor structure and function a monoclonal antibody plays a crucial role, thus providing useful tools for the study of receptor. However, because of the minute quantity of receptor molecules which could be obtained from natural sources, the generation of specific monoclonal antibody against receptor molecules from the purified receptors has been regarded as virtually impractical in consideration of cost and experimental times. The purpose of the present study was to generate and characterize a monoclonal antibody against human ${\beta}$2-adrenergic receptor. For the production of antibody, C-terminal regions of the human ${\beta}$2-adrenergic receptor was produced as a fusion protein with Glutathion S-transferase (GST) in E. Coli. The expression of the fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein was purified to an apparent homogeniety by affinity chromatography with Glutathion Sepharose CL-4B and used as an antigen for the immunization of BALB/C mice. The Production of monoclonal antibody was achieved by fusion of the immunized spleen cells and SP/2-0 myeloma cells. Positive hybridomas were screened by ELISA and were cloned by two consecutive rounds of limiting dilution. The monoclonal antibody produced in this study (mAb${\beta}$C02) was IgM type and purified by immunoaffinity chromatography using anti-mouse IgM agarose as an affinity matrix. MAb${\beta}$C02 showed strong and specific immunoreactivity against both the fusion protein and human ${\beta}$2-adrenergic receptor in ELISA and Western blot. The molecular weight of immunoreactive band was 64 kDa and exactly coincided with the previously reported molecular weight of ${\beta}$2-adrenergic recepters. The results of the present study suggest that mAb${\beta}$C02 may be used for the study of receptor function and regulation in normal or nonphysiological status.

• Journal of Microbiology and Biotechnology
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• v.24 no.6
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• pp.748-755
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• 2014

In the genome annotation of Escherichia coli MG1655, the orf382 (1,149 bp) is designated as a gene encoding an alcohol dehydrogenase that may be Fe-dependent. In this study, the gene was amplified from the genome by PCR and overexpressed in Escherichia coli BL21(DE3). The recombinant $6{\times}$His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates (n-butanol, $\small{L}$-threonine, ethanol, isopropanol, glucose, glycerol, $\small{L}$-serine, lactic acid, citric acid, methanol, or $\small{D}$-threonine), the enzyme showed the highest activity on $\small{L}$-threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of $\small{L}$-threonine into glycine. Considering the presence of tested substrates in living E. coli cel ls and previous literature, we believed that the suitable nomenclature for the enzyme should be an $\small{L}$-threonine dehydrogenase (LTDH). When using $\small{L}$-threonine as the substrate, the enzyme exhibited the best catalytic performance at $39^{\circ}C$ and pH 9.8 with $NAD^+$ as the cofactor. The determination of the Km values towards $\small{L}$-threonine (Km = $11.29{\mu}M$), ethanol ($222.5{\mu}M$), and n-butanol ($8.02{\mu}M$) also confirmed the enzyme as an LTDH. Furthermore, the LTDH was shown to be an ion-containing protein based on inductively coupled plasma-atomic emission spectrometry with an isoelectronic point of pH 5.4. Moreover, a circular dichroism analysis revealed that the metal ion was structurally and enzymatically essential, as its deprivation remarkably changed the ${\alpha}$-helix percentage (from 12.6% to 6.3%).

• Analytical Science and Technology
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• v.11 no.5
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• pp.374-385
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• 1998

Phytoestrogens are biologically active compounds derived from plants foods. It had been suggested that phytoestrogens, by inhibiting aromatase in peripheral and/or cancer cells and lowering estrogen levels, may play a protective role as antipromotional compounds during growth of estrogen-dependent cancers. Therefore, simultaneous analysis of estrogens and phytoestrogens is necessary to elucidate the possible involvement of phytoestrogens in estrogen metabolism. In this view, we developed a simple and reproducible procedure to quantitatively determine estrogen and phytoestrogen metabolites. The proposed method consisted of solid phase extraction using preconditioned Serdolit AD-2 resin, enzyme hydrolysis with ${\beta}$-glucuronidase/arylsulfatase from Helix pomatia, liquid-liquid extraction and TMS-ether derivatization. And the final determination was carried out by gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring mode (SIM). The precision and accuracy of this method was evaluated through within-a-day and day-to-day test. Recovery range and detection limit were 71.96~105.66%, 2~4 ng/mL, respectively. Using this method, 17 estrogen and 5 phytoestrogen compositions in urine of normal subjects were analyzed. It was found that amounts and relative distribution of urinary phytoestrigens and estrogens showed different pattern in male and female subjects.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.2
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• pp.244-251
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• 1997

The relationship between structure and biological activity was studied on the three neuropeptides (mammalian, trout- and goldfish-neuropeptide $\gamma$) that were syntheized by the solid-phase method. Circular dichroism spectra showed that mammalian, trout- and goldfish-neuropeptide $\gamma$ adopted an unordered structure in buffer solution. In the-presence of neutral and acidic liposomes, mammalian and trout-neuropeptioe $\gamma$ also took a random structure. However, goldfish-neuropeptide $\gamma$ took an $\alpha-helical$ structure in acidic liposomes. The intestinal motility response was investigated with carp intestines, guinea-pig ileums and rat duodenums. In case of carp intestine, contractile activity was as follows : goldfish-neuropeptide $\gamma\simeq$ trout-neuropeptide $\gamma>$ mammalian-neuropeptide $\gamma$, On the other hand, the contractile activity of mammalian-neuropeptide $\gamma$ was more potent than trout- and goldfish-neuropeptide $\gamma$ in the guinea-pig ileums and rat duodenums. These results suggest that neuropeptide $\gamma$ show the species-specific activity.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.1
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• pp.61-70
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• 2009

We have examined the effects of certain mutations of the selectivity filter and of the membrane helix M2 on $Ba^{2+}$ blockage of the inward rectifier potassium channel, Kir 2.1. We expressed mutant and wild type murine Kir 2.1 in Chinese hamster ovary(CHO) cells and used the whole cell patch-clamp technique to record $K^+$ currents in the absence and presence of externally applied $Ba^{2+}$. Wild type Kir2.1 was blocked by externally applied $Ba^{2+}$ in a voltage and concentration dependent manner. Mutants of Y145 in the selectivity filter showed little change in the kinetics of $Ba^{2+}$ blockage. The estimated $K_d(0)$ was 108 ${\mu}M$ for Kir2.1 wild type, 124 ${\mu}M$ for a concatameric WT-Y145V dimer, 109 ${\mu}M$ for a WT-Y145L dimer, and 267 ${\mu}M$ for Y145F. Mutant channels T141A and S165L exhibit a reduced affinity together with a large reduction in the rate of blockage. In S165L, blockage proceeds with a double exponential time course, suggestive of more than one blocking site. The double mutation T141A/S165L dramatically reduced affinity for $Ba^{2+}$, also showing two components with very different time courses. Mutants D172K and D172R(lining the central, aqueous cavity of the channel) showed both a decreased affinity to $Ba^{2+}$ and a decrease in the on transition rate constant(${\kappa}_{on}$). These results imply that residues stabilising the cytoplasmic end of the selectivity filter(T141, S165) and in the central cavity(D172) are major determinants of high affinity $Ba^{2+}$ blockage in Kir 2.1.

• Bulletin of the Korean Chemical Society
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• v.32 no.8
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• pp.2577-2583
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• 2011

We have designed a 20-residue hybrid peptide CA(1-8)-MA(1-12) (CAMA) incorporating residues 1-8 of cecropin A (CA) and residues 1-12 of magainin 2 (MA) with high bacterial cell selectivity. CAMA-P2 is an ${\alpha}$-helical antimicrobial peptide designed from a CAMA hybrid peptide and substitution of Gly-Ile-Gly hinge sequence of CAMA to Pro influences the flexibility at central part of CAMA. Based on structure-activity relationships of CAMA peptides, to investigate the effects of the total positive charges on antimicrobial activity of CAMA-P2, the $Ser^{14}{\rightarrow}$Lys analogue (CAMA-syn1) was synthesized. The role of tryptophan at C-terminal ${\alpha}$-helix on its antimicrobial activity as well as synergistic activity was also investigated using $Ser^{14}{\rightarrow}$Lys/$Phe^{18}{\rightarrow}$Trp analogue (CAMA-syn2). Also, we designed CAMA-syn3 by substitution of $Lys^{16}$ located opposite side of substituted $Lys^{14}$ of CAMA-syn1 with Leu residue, resulting in increase of hydrophobicity and amphipathicity of the peptide. All of CAMA-syn analogues showed good antimicrobial activities similar to those of CAMA and CAMA-P2. The CAMA-syn1 and CAMA-syn2 showed low hemolytic activity and cytotoxicity against human keratinocyte Haca-T cells while CAMA-syn3 showed hemolytic activity and cytotoxicity at its MIC value. We then investigated their abilities to act synergistically in combination with the antimicrobial flavonoids and synthetic compounds screened in our laboratory. The results showed that all peptides exhibited synergistic effects with dihydrobinetin, while only CAMA-syn2 exhibited synergistic effects with YKAs3001 against both S. aureus and MRSA, suggesting that Trp residue at C-terminus of CAMA-syn2 may facilitate the polar antibiotic flavonoids and synthetic compounds to permeabilize the membrane. This study will be useful for the development of new antibiotic peptides with potent antimicrobial and synergistic activity but without cytotoxicity.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.4
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• pp.1589-1595
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• 2014

Objective: Forkhead box C2 (FOXC2) is a member of the winged helix/forkhead box (Fox) family of transcription factors. It has been suggested to regulate tumor vasculature, growth, invasion and metastasis, although it has not been studied in cervical cancer. Here, we analyzed FOXC2 expression in cervical tissues corresponding to different stages of cervical cancer development and examined its correlation with clinicopathological characteristics. In addition, we examined the effects of targeting FOXC2 on the biological behavior of human cervical cancer cells. Methods: The expression of FOXC2 in normal human cervix, CIN I-III and cervical cancer was examined by immunohistochemistry and compared among the three groups and between cervical cancers with different pathological subtypes. Endogenous expression of FOXC2 was transiently knocked down in human Hela and SiHa cervical cells by siRNA, and cell viability and migration were examined by scratch and CCK8 assays, respectively. Results: In normal cervical tissue the frequency of positive staining was 25% (10/40 cases), with a staining intensity (PI) of $0.297{\pm}0.520$, in CIN was 65% (26/40cases), with a PI of $3.00{\pm}3.29$, and in cancer was 91.8% (68/74 cases), with a PI of $5.568 {\pm}3.449$. The frequency was 100% in adenocarcinoma (5/5 cases) and 91.3% in SCCs (63/69 cases). The FOXC2 positive expression rate was 88.5% in patients with cervical SCC stage I and 100% in stage II, showing significant differences compared with normal cervix and CIN. With age, pathologic differentiation degree and tumor size, FOXC2 expression showed no significant variation. On transient transfection of Hela and SiHa cells, FOXC2-siRNA inhibition rates were 76.2% and 75.7%; CCK8 results showed reduced proliferation and relative migration (in Hela cells from $64.5{\pm}3.16$ to $49.5{\pm}9.24$ and in SiHa cells from $60.1{\pm}3.05$ to $44.3{\pm}3.98$) (P < 0.05). Conclusion: FOXC2 gene expression increases with malignancy, especially with blood vessel hyperplasia and invasion degree. Targeted silencing was associated with reduced cell proliferation as well as invasion potential.

• Journal of Contemporary Eastern Asia
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• v.15 no.1
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• pp.41-63
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• 2016

Energy policy is known to have higher path dependency among policy fields (Kuper and van Soest, 2003; OECD, 2012; Kikkawa, 2013) and is a critical component of the infrastructure development undertaken in the early stages of nation building. Actor roles, such as those played by interest groups, are firmly formed, making it unlikely that institutional change can be implemented. In resource-challenged Japan, energy policy is an especially critical policy area for the Japanese government. In comparing energy policy making in Japan and Germany, Japan’s policy community is relatively firm (Hartwig et al., 2015), and it is improbable that institutional change can occur. The Japanese government’s approach to energy policy has shifted incrementally in the past half century, with the most recent being the 2012 implementation of the “Feed-In Tariff Law” (Act on Special Measures Concerning Procurement of Renewable Electric Energy by Operators of Electric Utilities), which encourages new investment in renewable electricity generation and promotes the use of renewable energy. Yet, who were the actors involved and the factors that influenced the establishment of this new law? This study attempts to assess the factors associated with implementing the law as well as the roles of the relevant major actors. In answering this question, we focus on identifying the policy networks among government, political parties, and interest groups, which suggests that success in persuading key economic groups could be a factor in promoting the law. Our data is based on the “Global Environmental Policy Network Survey 2012-2013 (GEPON2)” which was conducted immediately after the March 11, 2011 Great East Japan Earthquake with respondents including political parties, the government, interest groups, and civil society organizations. Our results suggest that the Feed in Tariff (FIT) Law’s network structure is similar to the information network and support network, and that the actors at the center of the network support the FIT Law. The strength of our research lays in our focus on political networks and their contributing mechanism to the law’s implementation through analysis of the political process. From an academic perspective, identifying the key actors and factors may be significant in explaining institutional change in policy areas with high path dependency. Close examination of this issue also has implications for a society that can promote renewable and sustainable energy resources.

• Korean Journal of Head & Neck Oncology
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• v.18 no.1
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• pp.11-17
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• 2002

Objectives: Expression of HMGI(Y), a nucleoprotein that binds to A/T rich sequences in the minor groove of the DNA helix, is observed in neoplastically transformed cells but not in normal cells. We have analyzed HMGI(Y), p53 expression and Ki-67 labelling index in squamous cell carcinomas of the head and neck, and evaluated its clinicopathologic significance. Materials and Methods: 40 cases of squamous cell carcinoma of the head and neck were entered on the study of immunohistochemical stains for HMGI(Y), p53 and Ki-67. We analyzed the relationship between HMGI(Y), p53, Ki-67 expression and age, sex, primary tumor site, stage, survival rate, recurrence. Results: HMGI(Y) expression evidenced by immunohistochemical staining was observed in 35 of 40 (87.5%) squamous cell carcinoma of the head and neck. But no significant correlation was observed between HMGI(Y) expression and other clinical factors such as primary site, tumor stage, differenciation, cervical lymph node, metastasis, recurrence and immunohistochemical status of p53. The Ki-67 labelling index was significantly correlated with recurrence and HMGI(Y) expression (p<0.05). Conclusion: This results suggest the Ki-67 is a good prognostic factor and the HMGI(Y) expression plays some roles in carcinogenesis and cellular proliferation of squamous cell carcinoma of the head and neck. HMGI(Y) gene can be used as a cancer marker, the correlation between the gene expression and the prognosis of the cancer patient should be proved in the future studies.