• IMMUNE NETWORK
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• v.9 no.2
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• pp.58-63
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• 2009

Background: T cell immunoglobulin and mucin domain containing 3 protein (Tim-3) expressed on terminally differentiated Th1 cells plays a suppressive role in Th1-mediated immune responses. Recently, it has been shown that N-glycosylation affects the binding activity of the Tim-3-Ig fusion protein to its ligand, galectin-9, but the binding properties of non-glycosylated Tim-3 on $CD4^+CD25^+$T cells has not been fully examined. In this study, we produced recombinant Tim-3-Ig fusion proteins in different cellular sources and its N-glycosylation mutant forms to evaluate their binding activities to $CD4^+CD25^+$T cells. Methods: We isolated and cloned Tim-3 cDNA from BALB/C mouse splenocytes. Then, we constructed a mammalian expression vector and a prokaryotic expression vector for the Tim-3-Ig fusion protein. Using a site directed mutagenesis method, plasmid vectors for Tim-3-Ig N-glycosylation mutant expression were produced. The recombinant protein was purified by protein A sepharose column chromatography. The binding activity of Tim-3-Ig fusion protein to $CD4^+CD25^+$T cells was analyzed using flow cytometry. Results: We found that the nonglycosylated Tim-3-Ig fusion proteins expressed in bacteria bound to $CD4^+CD25^+$T cells similarly to the glycosylated Tim-3-Ig protein produced in CHO cells. Further, three N-glycosylation mutant forms (N53Q, N100Q, N53/100Q) of Tim-3-Ig showed similar binding activities to those of wild type glycosylated Tim-3-Ig. Conclusion: Our results suggest that N-glycosylation of Tim-3 may not affect its binding activity to ligands expressed on $CD4^+CD25^+$T cells.

• BMB Reports
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• v.34 no.6
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• pp.537-543
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• 2001

In the present study, an in vitro ELISA system to assess the interaction between Src homology (SH)2 domains and phosphotyrosine that contain peptides was established using purified GST-conjugated SH2 proteins and synthetic biotinylated phosphotyrosine that contain oligopeptides. The SH2 domains bound the relevant phosphopeptides that were immobilized in the streptavidin-coated microtiter plate in a highly specific and dose-dependent manner. The epidermal growth factor receptor (EGFR)-, T antigen (T Ag)-, and platelet-derived growth factor receptor (PDGFR)-derived phosphopeptides interacted with the growth factor receptor binding protein (Grb)2/SH2, Lck/SH2, and phosphatidyl inositol 3-kinase (PI3K) p85/SH2, respectively. No cross-reactions were observed. Competitive inhibition experiments showed that a short phosphopeptide of only four amino acids was long enough to determine the binding specificity. Optimal concentrations of the GST-SH2 fusion protein and phosphopeptide in this new ELISA system for screening the binding blockers were chosen at 2nM and 500nM, respectively. When two candidate compounds were tested in our ELISA system, they specifically inhibited the Lck/SH2 and/or p85/SH2 binding to the relevant phosphopeptides. Our results indicate that this ELISA system could be used as an easy screening method for the discovery of specific binding blockers of protein-protein interactions via SH2 domains.

• Analytical Science and Technology
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• v.25 no.1
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• pp.33-38
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• 2012

Despite the importance of chloride binding, it is very difficult to measure the binding capacity, in particular, for the concrete body in an existing structure: in fact, the measurement procedure for chloride binding is much influenced by the environmental condition such as temperature, fineness of sample and pore water extraction techniques. The present study concerns the quantification of the binding capacity of chloride ions in concrete using the X-ray diffraction (XRD) technique. Once the binding isotherm of chlorides was determined by the Langmuir isotherm, as a function of the W/C, curing age and binder type, the generation of bound chlorides (i.e. Friedel's salt) was simultaneously ensured by the XRD technique. The amount of bound chloride was then determined by analyzing the peak intensity for the bound chlorides in the XRD curve. It was found that an increase in the curing age and a decrease in the W/C resulted in an increase in the binding capacity.

• Journal of Veterinary Clinics
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• v.24 no.3
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• pp.305-307
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• 2007

Lactoferrin-binding proteins (LBP) has not been well characterized in Streptococcus uberis isolated from milk of bovine mastitis and to date this protein is considered to be an important virulence factor in Streptococcal mastitis. To determine the more efficient extraction method of LBP from four S. uberis strains, we used two different extraction methods (mutanolysin and sodium dodecyl sulfate) in this study. Bacterial proteins extracted were electrophoresed by 10% polyacrylamide gels in the presence of sodium deodecyl sulfate and gels were transferred onto nitrocellulose membrane. Rabbit anti-bovine lactoferrin antibody and HRP-conjugated donkey anti-rabbit IgG antibody were used to detect LBP. This Western blotting analysis demonstrates that extraction method with SDS extracted 110 kDa and 112 kDa LBPs more efficiently compared to the mutanolysin extraction method.

• BMB Reports
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• v.31 no.1
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• pp.101-105
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• 1998

Immunoassay of botulinum toxin (BTX) B type was investigated using two typed of biosensors: light addressable potentiometric sensor (LAPS) and surface plasmon resonance (SPR) sensor. Urease-tagged and immuno-filtration capture method have been used for LAPS. Tag-free and direct binding real-time detection method have been used for SPR sensor. The detection limit of sandwich assay format with LAPS was 10 ng/ml, which was the lowest among methods tested. SPR has the advantage of being more convenient because tag-free direct binding assay can be used and reaction time was reduced, regardless of low sensitivity. This result shows that sandwich assay format with LAPS can be used as an alternative method of BTX mouse bioassay which is known as the most sensitive method for the detection of BTX.

• Journal of the Semiconductor & Display Technology
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• v.15 no.4
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• pp.92-96
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• 2016

Although deregistration attack is caused by simple spoofing the REGISTER message of a legitimate SIP UA, its impact is serious. The root cause of this attack is based on the fact that RFC 3261 allows the UA to remove the binding from the Location Server. In this paper, we propose a simple protection method to allow Registrar or Location Server just to ignore deregistration messages. We also show that this method works well by analyzing the process of registration and deregistration. Without any additional overhead such as an encryption or authentication, this method is able to establish a secure SIP environment efficiently protecting against the deregistration attack.

• Journal of the Korea institute for structural maintenance and inspection
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• v.12 no.5
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• pp.133-142
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• 2008

This paper studies the early-aged chloride binding capacity of various blended concretes including OPC(ordinary Portland cement), PFA(pulversied fly ash), GGBFS(ground granulated blast furnace slag) and SF(silica fume) cement paste. Cement pastes with 0.4 of a free water/binder ratio were cast with chloride admixed in mixing water, which ranged from 0.1 to 3.0% by weight of cement and different replacement ratios for the PFA, GGBFS and SF were used. The content of chloride in each paste was measured using water extraction method after 7 days curing. It was found that the chloride binding capacity strongly depends on binder type, replacement ratio and total chloride content. An increase in total chloride results in a decrease in the chloride binding, because of the restriction of the binding capacity of cement matrix. For the pastes containing maximum level of PFA(30%) and GGBFS(60%) replacement in this study, the chloride binding capacity was lower than those of OPC paste, and an increase in SF resulted in decreased chloride binding, which are ascribed to a latent hydration of pozzolanic materials and a fall in the pH of the pore solution, respectively. The chloride binding capacity at 7 days shows that the order of the resistance to chloride-induced corrosion is 30%PFA > 10%SF > 60%GGBFS > OPC, when chlorides are internally intruded in concrete. In addition, it is found that the binding behaviour of all binders are well described by both the Langmuir and Freundlich isotherms.

• The Korean Journal of Physiology
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• v.14 no.1
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• pp.7-13
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• 1980

In the previous experiment, authors have shown that during the latter half of estrous cycle there was an increase in plasma testosterone level in the rats stimulated with hCG. To determine the physiologic significance of elevated plasma testosterone, changes of the plasma concentrations of TeBG and testosterone following hCG stimulation were analyzed in the rats having a regular 5 day cycle. The rats were divided into three groups; the control, the rats stimulated with single hCG on the day of proestrus and stimulated with hCG throughout the entire cycle. Blood samples were obtained once a day for an estrous cycle and analyzed for the binding capacity of TeBG using ammonium sulphate precipitation method and testosterone concentration by means of radioimmunoassay. Followings were the results; 1) There was no significant variation in the binding capacity of TeBG in peripheral blood during the estrous cycle of the control rats. 2) No cyclic variation in the binding capacity of TeBG was observed in the rats stimulated with single hCG on proestrus. although the levels tended to be higher in the rats with stimulation than in the control rats. 3) Continual stimulation of hCG produced a marked increase in the binding capacity of TeBG especially on the day of metaestrus. 4) The changes in the plasma level of testosterone followed the same basic pattern seen in the TeBG binding capacity. 5) From above results, the followings were suggested. a. hCG related increase of the binding capacity of TeBG is probably secondary to a modest increase in estrogen as well. b. hCG related increase of plasma testosterone in female rats is not entirely due to excess production rather in part due to decreased metabolism induced by the rise in TeBG. c. It seems likely that most of elevated testosterone shown in the rat stimulated with hCG is bound to TeBG and only small portion is unbound form which influence cellular activity. It is rather possible that an increase in TeBG could augment estrogen activity.

• Journal of Applied Biological Chemistry
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• v.55 no.4
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• pp.263-266
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• 2012

Isolation of iron and calcium-binding peptides derived from cottonseed meal protein (CMP) hydrolysates was investigated. The degree of hydrolysis of CMP by Flavourzyme was monitored using trinitrobenzenesulfonic acid method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Enzymatic hydrolysis of CMP for 12 h was sufficient for the preparation of CMP hydrolysates, and the hydrolysates were membrane-filtered under 3 kDa as a molecular weight. The filtered solution was fractionated using Q-Sepharose fast flow, Sephadex G-15, and reversed phase-high performance liquid chromatography for iron and calcium-binding peptides. As a result, F51 fraction was obtained as the best candidate for calcium and iron chelation, and the isolated iron and calcium-binding peptides can be used as functional food additives, similar to iron and calcium supplements.

• Journal of Fashion Business
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• v.19 no.4
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• pp.57-74
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• 2015

The history of foot-binding was once regarded as a unique practice accentuating the beauty of a woman and the culture of the costume, to be found nowhere except in China. This thesis makes it an aim to understand the culture of costume in China by analyzing the beauty of foot-binding, and its meanings associated with the historical background of the costume. The method of investigation was by both previous literature on the topic and case analysis. The shoes were analyzed by colors, patterns and decorations; selecting 70 pieces easily accessible in the Tianjin Huaxia Shoes Culture Museum. In terms of form, the general type was of the most frequent, followed by one with an elevated toe, mixed type, and one of the downward toe, sequentially. The color of foot-binding shoes were mostly vivid chromatic colors, while the achromatic ones like black and grey constituting significant portions. In view of the patterns, plants patterns were the most frequent, followed by the mixed patterns of mostly flowers with butterflies, bats, and birds desiring the prosperity of descendants. In view of the ornaments, embroidery was the most frequent, while the mixed shoes included tassels, sequins, applique, and shoes without ornaments were of distributions. The forms, colors, patterns and ornaments clearly associated with the wishes of artistic beauty, sexuality, and rise in women's social status may be in the hope of leading others to understand the costume culture embedded in the Chinese by providing important clues about them.