• BMB Reports
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• v.51 no.5
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• pp.207-208
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• 2018

Chitinase-Like Proteins (CLPs) are an evolutionarily conserved protein which lose their enzymatic activity for degrading chitin macromolecules. Chitinase-3-like-1 (Chi3l1) is a type of CLP that is highly expressed in epithelial cells, macrophages, etc., and is known to have correlations with type 2 inflammation and cancer. Although the increased level of Chi3l1 in the blood was reported in various disease patients, the function of Chi3l1 in adaptive immunity has been totally unknown. Recently, we found that Chi3l1 is expressed in T cells and has a negative regulatory role in T-cell activation and proliferation. A genetic ablation study of Chi3l1 in T cells showed hyperresponsiveness to TcR stimulation, which increased proliferation and Th1 differentiation. A significant increase of $IFN{\gamma}$ signaling in Chi3l1-deficient T cells synergistically increased Th1 and CTL functions against melanoma cells in vitro and in vivo. In addition, targeted knockdown by Chi3l1 siRNA complexed with the cell-penetrating peptide dNP2, which showed decreased pulmonary melanoma metastasis with increased infiltration of Th1 and CTL in the lung. This study first suggests that Chi3l1 is a novel regulator of Th1/CTL responses and could be a target for treating cancer to increase tumor immunity.

• Journal of Microbiology and Biotechnology
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• v.19 no.10
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• pp.1169-1175
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• 2009

An antifungal chitinase, ChiCW, produced by Bacillus cereus 28-9 is effective against conidial germination of Botrytis elliptica, the causal agent of lily leaf blight. ChiCW as a modular enzyme consists of a signal peptide, a catalytic domain, a fibronectin type-III-like domain, and a chitin-binding domain. When two C-terminal domains of ChiCW were truncated, $ChiCW{\Delta}FC$ (lacking the chitin-binding domain and fibronectin type III-like domain) lost its antifungal activity. Since $ChiCW{\Delta}C$ (lacking the chitin-binding domain) could not be expressed in Escherichia coli as $ChiCW{\Delta}FC$ did, a different strategy based on protein engineering technology was designed to investigate the involvement of the chitin-binding domain of ChiCW ($ChBD_{ChiCW}$) in antifungal activity in this study. Because ChiA1 of Bacillus circulans WL-12 is a modular enzyme with a higher hydrolytic activity than ChiCW but not inhibitory to conidial germination of Bo. elliptica and the similar domain composition of ChiA1 and ChiCW, the C-terminal truncated derivatives of ChiA1 were generated and used to construct chimeric chitinases with $ChBD_{ChiCW}$. When the chitin-binding domain of ChiA1 was replaced with $ChBD_{ChiCW}$, the chimeric chitinase named ChiAAAW exhibited both high enzyme activity and antifungal activity. The results indicate that $ChBD_{ChiCW}$ may play an important role in the antifungal activity of ChiCW.

• Horticultural Science & Technology
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• v.31 no.3
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• pp.275-281
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• 2013

Low temperature is one of the major environmental factors that affect productivity including reduced growth and budding of vines, and changes of metabolic processes in grape (Vitis spp.). To screen the specific expression of abiotic stress-related genes against cold treatment in 'Kyoho' and 'Campbell Early' grapevines, expression of various defense-related genes was investigated by RT-PCR and real-time PCR. Among the 67 genes analyzed by RT-PCR and real-time PCR, 17 and 16 types of cDNA were up-regulated, while 5 and 6 types were down-regulated in cold-treated 'Kyoho' and 'Campbell Early' grapevines, respectively. Genes encoding carotene (Cart3564 and Cart4472), chalcone isomerase (CHI), cytochrome P450 (CYP), flavonol synthase (FLS), endo-${\beta}$-glucanase precursor (Glu), glutathione peroxidase (GPX), glutathione-S-transferase (GST), leucine-rich repeats (LRR), manganese superoxide dismutase (Mn-SOD), phenylalanine ammonia lyase (PAL), polygalacturonase-inhibiting protein (PGIP), proline rich protein 2 (PRP2), small heat shock protein (sHSP), temperature induced lipocalin (TIL), and thaumatin-like protein (TLP) were up-regulated, while those encoding CBF like transcription factor (CBF1), chitinase-like protein (CLP), cold induced protein (CIP), glycerol-3-phosphate acyltransferase (GPAT), and mitogen-activated protein kinase (MAPK) were down-regulated by low temperature treatment in both in 'Kyoho' and 'Campbell Early'.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.48 no.5
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• pp.407-412
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• 2003

In order to measure an antifreezing tolerance, antifreeze proteins accumulated upon cold acclimation in apoplast were analyzed. As Dongborilho were cold-acclimated for 3 to 74 days there was an abrupt increase in apoplastic proteins up to 30 days and then decrease to the similar levels. Among the known antifreeze proteins, CLP produced in E. coli. and TLP purified from apoplast were used to generate antisera that allow to measure and localize the proteins in leaves of barley. The CLP of 27.7 kDa and TLPs of 6, 26, 27, 30, and 31 kDa were increased in their amounts in apoplast as cold treatment being longer. There was a correlation among the amounts of those proteins accumulated in apoplast and freezing tolerance as shown in field and ion leakage tests for five cultivars. The deposit of CLP was localized in the marginal area and the area adjacent to leaf vescular bundle cells in an increasing manner according to duration of cold acclimation but no variation was observed in terms of it's distribution. Based on the close correlation between levels of antifreeze proteins and degrees of freezing tolerance, the immunological methods was to develop to estimate a freezing tolerance in barley.

• IMMUNE NETWORK
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• v.21 no.3
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• pp.22.1-22.17
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• 2021

Chitinase-3-like-1 (CHI3L1) is known to induce inflammation in the progression of allergic diseases. Previous our studies revealed that 2-({3-[2-(1-cyclohexen-1-yl)ethyl]-6,7-dimethoxy-4-oxo-3,4-dihydro-2-quinazolinyl}sulfanyl)-N-(4-ethylphenyl)butanamide (K284-6111; K284), the CHI3L1 inhibiting compound, has the anti-inflammatory effect on neuroinflammation. In this study, we investigated that K284 treatment could inhibit the development of atopic dermatitis (AD). To identify the effect of K284, we used phthalic anhydride (5% PA)-induced AD animal model and in vitro reconstructed human skin model. We analyzed the expression of AD-related cytokine mediators and NF-κB signaling by Western blotting, ELISA and quantitative real-time PCR. Histological analysis showed that K284 treatment suppressed PA-induced epidermal thickening and infiltration of mast cells. K284 treatment also reduced PA-induced release of inflammatory cytokines. In addition, K284 treatment inhibited the expression of NF-κB activity in PA-treated skin tissues and TNF-α and IFN-γ-treated HaCaT cells. Protein-association network analysis indicated that CHI3L1 is associated with lactoferrin (LTF). LTF was elevated in PA-treated skin tissues and TNF-α and IFN-γ-induced HaCaT cells. However, this expression was reduced by K284 treatment. Knockdown of LTF decreased the expression of inflammatory cytokines in TNF-α and IFN-γ-induced HaCaT cells. Moreover, anti-LTF antibody treatment alleviated AD development in PA-induced AD model. Our data demonstrate that CHI3L1 targeting K284 reduces AD-like skin inflammation and K284 could be a promising therapeutic agent for AD by inhibition of LTF expression.

• The Plant Pathology Journal
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• v.35 no.5
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• pp.530-537
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• 2019

Fuji, a major apple cultivar in Korea, is susceptible to white rot. Apple white rot disease appears on the stem and fruit; the development of which deteriorates fruit quality, resulting in decreases in farmers' income. Thus, it is necessary to characterize molecular markers related to apple white rot resistance. In this study, we screened for differentially expressed genes between uninfected apple fruits and those infected with Botryosphaeria dothidea, the fungal pathogen that causes white rot. Antimicrobial tests suggest that a gene expression involved in the synthesis of the substance inhibiting the growth of B. dothidea in apples was induced by pathogen infection. We identified seven transcripts induced by the infection. The seven transcripts were homologous to genes encoding a flavonoid glucosyltransferase, a metallothionein-like protein, a senescence-induced protein, a chitinase, a wound-induced protein, and proteins of unknown function. These genes have functions related to responses to environmental stresses, including pathogen infections. Our results can be useful for the development of molecular markers for early detection of the disease or for use in breeding white rotresistant cultivars.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.43 no.3
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• pp.172-178
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• 1998

Freezing-resistant plants can survive subzero temperatures by withstanding extracellular ice formation. During cold acclimation, their leaves accumulate antifreeze proteins (AFPs) that are secreted into the apoplast and have the ability to modify the normal growth of ice crystals. Three barley, two wheat and two rye cultivars were grown under two different temperature regimes (20/16$^{\circ}C$ and 5/2$^{\circ}C$, day/night). Apoplastic proteins from winter cereals were separated by SDS-PAGE and detected with antisera to AFPs from winter rye. Apoplastic proteins accumulated to much higher levels in cold-acclimated (CA) leaves compared with nonacclimated (NA) ones in winter cereals. After cold acclimation, the protein concentration of apoplastic extracts increased significantly from 0.088 $mgmL^{-1}$ to 0.448 $mgmL^{-1}$, with about 5-fold increment. Also, the apoplastic protein content per gram leaf fresh weight in CA leaves ranged from 31 $\mu\textrm{g}$ $(gFW)^{-1}$ to 120 $\mu\textrm{g}$ $(gFW)^{-1}$ with an averaged value of 77 $\mu\textrm{g}$ $(gFW)^{-1}$, and coefficients of variation of 54.9%. The CA leaves in Musketeer (a Canadian winter rye cultivar) showed the greatest AFPs and antifreeze activity followed by 'Geurumil' (a Korean winter wheat cultivar), and 'Dongbori l' (Korean facultative barley cultivar). The proteins secreted into the wheat leaf apoplast at CA condition were more numerous than those observed in winter rye, where two $\beta$-1,3-glucanase-like proteins (GLPs), two chitinase-like proteins (CLPs) and two thaumatin-like proteins (TLPs) accumulated during cold acclimation. The proteins in barley leaf apoplast at CA conditions were a little different from those in wheat leaves. The AFPs were various among and within species. More freezing-resistant cultivars had more clear and numerous bands than less freezing-resistant ones. The high determination coefficient ($R^2$ =91 %) between freezing resistance and AFPs per gram leaf fresh weight indicated that the amount of AFPs was highly related to freezing resistance in winter cereal crops.

• Journal of Plant Biotechnology
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• v.30 no.4
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• pp.315-321
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• 2003

Freezing and drought tolerances in plants are very important for survival in the desert. In an effort to reduce desertifcation in Gobi, a molecular breeding of Artemisia adamsii using the CLP (chitinase like protein, antifreeze protein) and Dhn5 (dehydrin5) genes from barley is performed by introducing them into Artemisia adamsii via Agrobacteria. We had found an optimal combinatorial concentration of hormones at 0.05mg/L of NAA and 0.5mg/L of BA for growth of callus in Artemisia adamsii. In addition, the higher rate of callus induction using hypocotyl as explant was observed comparing to explants of stem and leaf. There were some variations in the level of the proteins expressed among the transgenic lines such that the lines of CLP(CS1-5, 1-7,4-4) and Dhn5(DS2-2, 2-3) lines produce the protein to higher levels. The transgenic lines showing a higher level of Dhn5 exhibited better growth than nontransgenic callus in presence of 10 and 20％ PEG. In case of the CLP tansgenic lines, both CS1-5 and CS1-7 showed a higher level of freezing tolerance determined by ion leakage test.

• Journal of Microbiology and Biotechnology
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• v.11 no.2
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• pp.337-341
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• 2001

To isolate antifungal substances from Korean radish (Raphanus Sativus L.) seeds, various purification techniques such as DE52 cellulose anion exchange, SP-Sephadex C-25 cation exchange, and Sephadex G-50 gel filtration chromatographies were used. The molecular masses of two purified R. sativus antifungal proteins (RAPs) were estimated to be about 6.1 kDa (RAP-1) and 6.2 kDa (RAP-2) by SDS-PAGE, and 5.8 kDa(RAP-1) and 6.2 kDa (RAP-2 by a gel filtration chromatography, respectively. Purified proteins RAP-1 and 2 clearly exhibited different growth inhibitory activities against other microorganisms like Candida albicans and Saccharomyces cerevisiae. Although they have similar molecular masses, both RAP-1 and 2 proteins are not identical because their microbial inhibitory actions were different. Therefore, RAP-1 could be a new antifungal protein when compared with the antifungal activities of 2S albumins, Rs-AFPs, Mj-AMPs, chitinase, glucanase, permatin, and ribosome inactivating proteins, all of which are anifungal proteins of plants.

• The Plant Pathology Journal
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• v.30 no.4
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• pp.343-354
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• 2014

Rice blast disease caused by Magnaporthe oryzae is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, we utilized the transcriptome analysis approach using a 300 K tilling microarray to rice leaves infected with compatible and incompatible M. oryzae strains. Prior to the microarray experiment, total RNA was validated by measuring the differential expression of rice defense-related marker genes (chitinase 2, barwin, PBZ1, and PR-10) by RT-PCR, and phytoalexins (sakuranetin and momilactone A) with HPLC. Microarray analysis revealed that 231 genes were up-regulated (>2 fold change, p < 0.05) in the incompatible interaction compared to the compatible one. Highly expressed genes were functionally characterized into metabolic processes and oxidation-reduction categories. The oxidative stress response was induced in both early and later infection stages. Biotic stress overview from MapMan analysis revealed that the phytohormone ethylene as well as signaling molecules jasmonic acid and salicylic acid is important for defense gene regulation. WRKY and Myb transcription factors were also involved in signal transduction processes. Additionally, receptor-like kinases were more likely associated with the defense response, and their expression patterns were validated by RT-PCR. Our results suggest that candidate genes, including receptor-like protein kinases, may play a key role in disease resistance against M. oryzae attack.