• Asian Pacific Journal of Cancer Prevention
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• v.13 no.8
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• pp.4031-4036
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• 2012

Background: The negative signaling provided by interactions of the co-inhibitory molecule, programmed death-1 (PD-1), and its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), is a critical mechanism contributing to tumor evasion; blockade of this pathway has been proven to enhance cytotoxic activity and mediate antitumor therapy. Here we evaluated the anti-tumor efficacy of AAV-mediated delivery of the extracellular domain of murine PD-1 (sPD-1) to a tumor site. Material and Methods: An rAAV vector was constructed in which the expression of sPD-1, a known negative regulator of TCR signals, is driven by human cytomegalovirus immediate early promoter (CMV-P), using a triple plasmid transfection system. Tumor-bearing mice were then treated with the AAV/sPD1 construct and expression of sPD-1 in tumor tissues was determined by semi quantitative RT-PCR, and tumor weights and cytotoxic activity of splenocytes were measured. Results: Analysis of tumor homogenates revealed sPD-1 mRNA to be significantly overexpressed in rAAV/sPD-1 treated mice as compared with control levels. Its use for local gene therapy at the inoculation site of H22 hepatoma cells could inhibit tumor growth, also enhancing lysis of tumor cells by lymphocytes stimulated specifically with an antigen. In addition, PD-1 was also found expressed on the surfaces of activated CD8+ T cells. Conclusion: This study confirmed that expression of the soluble extracellular domain of PD-1 molecule could reduce tumor microenvironment inhibitory effects on T cells and enhance cytotoxicity. This suggests that it might be a potential target for development of therapies to augment T-cell responses in patients with malignancies.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.4
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• pp.1603-1609
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• 2014

Background: The response to treatment and overall survival (OS) of patients with acute myeloid leukemia (AML) is variable, with a median ranging from 6 months to 11.5 years. TP53 is associated with old age, chemotherapy resistance, and worse OS. Using genetic sequencing, we set out to look at our own experience with AML, and hypothesized that both TP53 mutations and SNPs at codon 72 would mimic the literature by occurring in a minority of patients, and conferring a worse OS. Materials and Methods: We performed a pilot study of randomly selected, newly diagnosed AML patients at Mount Sinai Medical Center, diagnosed from 2005-2008 (n=10). TP53 PCR sequencing was performed using DNA from bone marrow smears. Analysis was accomplished using Mutation Surveyor software with confirmation of the variants using the COSMIC and dbSNP databases. Results: Fewer than half of the patients harbored TP53 mutations (40%). There was no significant difference in OS based on gender, AML history, risk-stratified karyotype, or TP53 mutation. There were possible trends toward improved survival among patients less than 60 (11 vs 4 months, p=0.09), Hispanics (8 vs 1 months, p=0.11), and those not harboring SNP P72R (8 vs 2 months, p=0.10). There was a significant improvement in survival among patients with better performance status (28 vs 4 months, p=0.01) and those who did not have a complex karyotype (8 vs 1 months, p=0.03). The most commonly observed TP53 mutation was a missense N310K (40%) and the most commonly observed SNP was P72R (100.0%). Conclusions: Our study confirms previous reports that poor PS and the presence of a complex karyotype are associated with a decreased OS. In our cohort, TP53 mutations were relatively common, occurring more frequently in male patients with an adverse karyotype. Although there was no significant difference in survival between TP53 mutated and un-mutated patients, there was a possible trend toward worse OS among patients with SNP P72R. Larger studies are needed to validate these findings.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.18
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• pp.7965-7970
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• 2014

One of the goals of tumor immunotherapy is to generate immune cells with potent anti-tumor activity through in vitro techniques using peripheral blood collected from patients. However, cancer patients generally have poor immunological function. Thus using patient T cells, which have reduced in vitro proliferative capabilities and less tumor cell killing activity to generate lymphokine-activated killer (LAK) cells, fails to achieve optimal clinical efficacy. Interleukin-2 (IL-2) is a potent activating cytokine for both T cells and natural killer cells. Thus, this study aimed to identify optimal donors for allogeneic LAK cell immunotherapy based on single nucleotide polymorphisms (SNP) in the IL-2 and IL-2R genes. IL-2 and IL-2R SNPs were analyzed using HRM-PCR. LAK cells were derived from peripheral blood mononuclear cells by culturing with IL-2. The frequency and tumor-killing activity of LAK cells in each group were analyzed by flow cytometry and tumor cell killing assays, respectively. Regarding polymorphisms at IL-2-330 (rs2069762) T/G, LAK cells from GG donors had significantly greater proliferation, tumor-killing activity, and IFN-${\gamma}$ production than LAK cells from TT donors (P<0.05). Regarding polymorphisms at IL-2R rs2104286 A/G, LAK cell proliferation and tumor cell killing were significantly greater in LAK cells from AA donors than GG donors (P<0.05). These data suggest that either IL-2-330(rs2069762)T/G GG donors or IL-2R rs2104286 A/G AA donors are excellent candidates for allogeneic LAK cell immunotherapy.

• Korean Journal of Ichthyology
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• v.29 no.1
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• pp.62-68
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• 2017

The Alaska pollock (Gadus chalcogrammus) belongs to the family Gadidae; it is a cold water fish, and has been developed as a novel aquaculture species in Korea. In this study, we describe ongoing surveillance for aquatic animal pathogens based on growth stages. We investigated bacterial flora in rearing water, and monitored pathogens; we also analyzed histopathological traits of abnormal fish. In rearing water, the total bacterial counts were $2.1{\times}10^3cfu/mL$ and Vibrio spp. (52%) were predominant in the larvae stage. In the juvenile and adult stages, the total bacterial counts were $3.4{\times}10^3$ and $3.2{\times}10^2cfu/mL$, respectively (with Pseudomonas sp. as the predominant species; 90% and 52%). This result revealed that the bacterial flora in rearing water changed depending on the feeding types. No virulent-bacteria or problematic viruses (VHSV, viral hemorrhagic septicemia virus; NNV, nervous necrosis virus; MBV, marine birnavirus) were detected from outwardly healthy fish using either culture or PCR assay. Some juveniles (less than 5%) had gas bubbles on the gill lamellae, degeneration of the corneal epithelium, and choroid gland degeneration, suggesting that these symptoms were caused by external injury and secondary infection by opportunistic bacteria. Disease management is important to cope with disease emergence in the novel aquaculture species Alaska pollock.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.98-98
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• 2002

Recruitment of primordial follicles(PMF) is crucial for female fertility. however, factors and mechanisms that regulate this process is poorly understood. The present study was conducted to obtain an inclusive view of the gene expression and to identify novel factors and their pathways of regulating PMF arrest and/or growth initiation. Ovaries from one-day neonatal(consists of oocyte and PMF) and five-day old(consists of PMF and primary follicles, PRIF) mice were collected, either total RNA or mRNA was isolated, and suppression subtractive hybridization(SSH) was used to isolate and clone genes that differentially expressed in day 1 and day 5 ovaries. Confirmation that some of these genes are differentially expressed in PMF and/or in PRIF was accomplished by using laser captured microdissection(LCM), RT-PCR. in situ hybridization(ISH) and/or immunohistochemistry(IHC). In toto, 357 clones were sequenced and analyzed by BLAST and RIKEN program. Sequences of 330 clones significantly matched database entries while 27 clones were novel. Forty-two and 47 different genes were identified as differentially expressed in day 1 and day 5 ovaries, respectively, while 7 genes were expressed in both stages of ovaries. Day 5-subtracted library included several genes known as markers far growing follicles, such as ZP2, MATER, and fetuin. Among the genes with assigned functions, 23.8% was associated with cell cycle/apoptosis regulation, 7.1% with cellular structure, 11.9% with metabolism, 26.2% with signal transduction, and 31.0% with gene/protein expression in day 1; while 10.6%, 17.0%, 23.5%, 25.5%, and 23.4% in day 5, respectively. Genes such as GDF-8, Lats2, Septin2, and Weel were the highly expressed genes in PMF, while HSP84, Laminin2, MATER, MTi7, PTP, and Wrn were highly expressed genes in PRIF. We have successfully discovered list of genes expressed in day 1 and day 5 ovaries and confirmed that some of them are differentially expressed in PMF and/or PRIF. Gene expression profile from the present study would provide insight for the future study on the mechanism(s) involved in primordial-primary follicular transition. This work was Supported by Korean Health 21 RND Project, Ministry of Health and Welfare, Korea (01-PJ10-PG6-01GN13-0002).

• Journal of Physiology & Pathology in Korean Medicine
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• v.31 no.6
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• pp.374-379
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• 2017

Hair loss affects interpersonal relationships and causes psychological stress. In this study, we investigated the antioxidant activity of Cynanchi Wilfordii Radix (CWR) and its effects on dermal papilla (DP) cells. Antioxidant efficacy was examined by ABTS assay. To confirm the effect on cell activity, MTS assay was performed and cell count was directly measured by hemocytometer. The mRNA expression of genes involved in hair formation and hair loss formation was confirmed by quantitative RT-PCR. CWR has a strong antioxidant activity. Cell viability of DP cells was increased to 118.5% by treatment of 0.5 mg/ml CWR for 24 hours, but the effect on the cell number was insignificant. These results suggest that CWR increases mitochondrial activity without promoting cell proliferation. Treatment of DP cells with 0.5 mg/ml CWR resulted in 48.5% reduction of mRNA expression of type 2 $5{\alpha}$-reductase, a major cause of male hair loss. In addition, mRNA expression of bone morphogenetic pretein (BMP), fibroblast growth factor (FGF)7, and FGF10, which are closely related to hair growth, was also decreased. Reactive oxygen species (ROS) acts as a cause of hair loss. The excellent antioxidant efficacy of CWR is thought to be able to effectively remove ROS. The dihydrotestosterone produced by type 2 $5{\alpha}$-reductase in DP cells is a potent inducer of male pattern hair loss. The inhibitory effect of type 2 $5{\alpha}$-reductase mRNA on DP cells induced by CWR may induce a positive therapeutic effect of male pattern hair loss.

• Journal of Plant Biotechnology
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• v.41 no.3
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• pp.159-167
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• 2014

In eukaryotes, proteins that are secreted into ER are post-translationally modified by N-glycosylation, the patterns of which are significantly different between plant and animal cells. Biotechnology industry has already produced a number of therapeutic glycoproteins in plant cells. However, the aberrant glycosylation of therapeutic recombinant proteins in plant systems can cause immune problems in humans. Therefore, it is important to develop strategies for producing non-glycosylated forms to preserve biological activity and native conformation by a peptide: N-glycanase (PNGase). In this study, we try to isolate PNGase T gene from tomato, which can use as a platform plant for biotechnology industry. We isolated a cDNA (GenBank Accession number KM401550) from tomato leaves with 1,767 bp, which encoded a polypeptide of 588 amino acids with a predicted molecular mass of 65.8 kDa. We also investigated the expression patterns of PNGase T during fruit ripening of tomato. The transcripts of PNGase T, which were constitutively induced in tomato fruit from green stage, were significantly increased and reached a peak at orange stage. After which, those transcripts were continuously reduced. The expression pattern of PNGase T was coincided well with transcripts profiles of metacaspase gene, LeMCA, and senescence-related gene members of ACC synthase, LeACS2, LeACS4, and LeACS6, for ethylene biosynthesis during fruit ripening. These results suggest that PNGase T is involved in a de-glycosylation process associated with senescence and fruit ripening.

• Journal of Plant Biotechnology
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• v.40 no.1
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• pp.37-42
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• 2013

Rice is one of the most important cereal crops as a model plant for functional genomics of monocotyledons and usually transformed using Agrobacterium tumefaciens. However, the transformation's process using previous method is still time consuming and uneconomical, low efficiency. In this study, we established a new method by modifying the general Agrobacterium protocol especially in the infection and co-cultivation, Agrobacterium elimination, infected calli's selection steps using liquid media. We directly inoculated Agrobacterium containing a ZjLsL gene under the control of constitutive promoter into the 1- to 3-week-old rice calli derived from mature seeds. After 3 days of co-cultivation, the infected calli were transferred onto liquid media of Agrobacterium elimination and calli's selection for 3 days. The calli were transferred to calli's growth solid media for 14 days and then the calli transferred to shoot induction and root induction media. Putative transformants were initially selected on the medium containing phosphinothricin, and the PAT protein verified by PAT strip test. This method in this study would lead to reduction of substantial labor and time to generate transgenic plants.

• Journal of Veterinary Clinics
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• v.31 no.1
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• pp.6-10
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• 2014

Siberian Roe deer which inhabits Jeju Island is unique native species. Most of all the roe deer infect a lot of ticks, which can affect its population directly and can act as a vector to spread vector-borne diseases. The purposes of this study were to identify the ticks and detect the piroplamsosis on the roe deer in Jeju island. We collected ticks and blood samples in 23 roe deer rescued and treated at the Jeju Wildlife Rescue Center. As a result, we identified the one species of ticks, Haemaphysalis longicornis in roe deer and detected the closely related to Theileria luwenshuni in all blood samples (100%) and 8 pooled ticks (34.8%). These results indicate that there may be a high prevalence particularly of T. luwenshuni infection in Jeju wild roe deer and H. longicornis is a major vector of these diseases. It suggested that Jeju roe deer may act as reservoirs for these zoonotic pathogens.

• Preventive Nutrition and Food Science
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• v.19 no.3
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• pp.136-144
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• 2014

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This study was conducted to evaluate the effects of Platycarya strobilacea S. et Z. (PSE) extract on mouse hair growth and to determine the mechanism of action of PSE. PSE was purchased and its antioxidant activities, such as electron donating ability, total polyphenol content, and flavonoid content were tested. Toxicity during topical treatment was determined by the CCK-8 assay, a cell viability test. Fifteen 4-week-old male C57BL/6 mice were assigned to receive one of three treatments: dimethyl sulfoxide (negative control), minoxidil (positive control) or PSE. Test materials were topically applied to the shaved dorsal skin of each mouse daily for 3 weeks. After 21 days, we observed skin tissue hair follicle morphology and length, mast cell number, and stem cell factor (SCF) expression using hematoxylin and eosin (H&E), toluidine blue, and immunohistochemical staining, respectively. Furthermore, the expression of cytokines involved in hair growth [i.e., insulin-like growth factor (IGF)-1, keratinocyte growth factor (KGF), and transforming growth factor (TGF)-${\beta}1$] was determined by PCR. PSE was found to have very high antioxidant activity. The cell viability rate of PSE-treated mice was markedly higher than that of mice in the control group. We also observed an increase in hair follicle length, strong SCF staining, and a decrease in mast cell number in the PSE group. In addition, PSE-treated mice had higher IGF-1 and KGF expression and lower TGF-${\beta}1$ expression than mice in the minoxidil-treated group. These results suggest that topical application of PSE promotes hair growth by intensifying SCF, suppressing mast cell production, and increasing hair growth-promoting cytokine expression.