• IMMUNE NETWORK
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• v.12 no.1
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• pp.33-39
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• 2012

Background: Therapeutic approaches using monoclonal antibodies (mAbs) against complement regulatory proteins (CRPs:i.e.,CD46,CD55 and CD59) have been reported for adjuvant cancer therapy. In this study, we generated a recombinant 1E8 single-chain anti-CD59 antibody (scFv-Fc) and tested anti-cancer effect.by using complement dependent cytotoxicity (CDC). Methods: We isolated mRNA from 1E8 hybridoma cells and amplified the variable regions of the heavy chain (VH) and light chain (VL) genes using reversetranscriptase polymerase chain reaction (RT-PCR). Using a linker, the amplified sequences for the heavy and light chains were each connected to the sequence for a single polypeptide chain that was designed to be expressed. The VL and VH fragments were cloned into the pOptiVEC-TOPO vector that contained the human CH2-CH3 fragment. Then, 293T cells were transfected with the 1E8 single-chain Fv-Fc (scFv-Fc) constructs. CD59 expression was evaluated in the prostate cancer cell lines using flow cytometry. The enhancement of CDC effect by mouse 1E8 and 1E8 scFv-Fc were evaluated using a cytotoxicity assay. Results: The scFv-Fc constructs were expressed by the transfected 293T cells and secreted into the culture medium. The immunoreactivity of the secreted scFv-Fc construct was similar to that of the mouse 1E8 for CCRF-CEM cells. The molecular masses of 1E8 scFv-Fc were about 120 kDa and 55 kDa under reducing and non-reducing conditions, respectively. The DNA sequence of 1E8 scFv-Fc was obtained and presented. CD59 was highly expressed by the prostate cancer cell line. The recombinant 1E8 scFv-Fc mAb revealed significantly enhanced CDC effect similar with mouse 1E8 for prostate cancer cells. Conclusion: A 1E8 scFv-Fc construct for adjuvant cancer therapy was developed.

• Animal cells and systems
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• v.16 no.2
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• pp.87-94
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• 2012

Nur77 is a member of the nuclear receptor 4A (NR4A) subgroup, which has been implicated in energy metabolism. Although Nur77 is found in adipose tissue, where TR4 plays a key role in lipid homeostasis, the role of Nur77 in adipogenesis is still controversial. Although the Nur77 responsive element (AAAGGTCA) is partially overlapped with TR4-binding sites (AGGTCA $n$ AGGTCA: $n$=0-6), the regulatory role of Nur77 in TR4 function associated with adipocyte biology remains unclear. Here, we found that Nur77 inhibits adipogenesis and TR4 transcriptional activity. Treatment with a Nur77 agonist, 1,1-bis(3'-indolyl)-1-($p$-anisyl)-methane, during 3T3-L1 adipocyte differentiation reduced adipogenesis. In reporter gene analysis, Nur77 specifically suppressed TR4 transcription activity but had little effect on $PPAR{\gamma}$ transcription activity. Consistently, Nur77 also suppressed TR4-induced promoter activity of the TR4 target gene PEPCK, which is known to be important for glyceroneogenesis in adipose tissue. Furthermore, Nur77 suppressed TR4 binding to TR4 response elements without direct interaction with TR4, suggesting that Nur77 may inhibit TR4 transcription activity via binding competition for TR4-binding sites. Furthermore, DIM-C-$pPhOCH_3$ substantially suppressed TR4-induced PEPCK expression in 3T3-L1 adipocytes. Together, our data demonstrate that Nur77 plays an inhibitory role in TR4-induced PEPCK expression in 3T3-L1 adipocytes.

• Journal of Periodontal and Implant Science
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• v.30 no.4
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• pp.885-894
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• 2000

Deer antler has been widely prescribed in Chinese and Korean pharmacology. Although there have been several reports concerning the effects of deer antler, such as anti-aging action, anti-inflammatory activity, antifungal action and regulatory activity of the level of glucose, the effect on bone has not determined yet. The purpose of this study was to examine the effect of deer antler on osteoblast differentiation. Hexane extract(CN-H) and chloroform extract(CN-C) were acquired from deer antler(Cervus nippon) and MC3T3-E1 pre-osteoblasts were cultured in the presence or absence of each extract. Osteoblast differentiation was estimated with the formation of mineralized nodules and the mRNA expression of alkaline phosphatase(ALP), osteocalcin(OC) and bone sialoprotein(BSP) which are markers of osteoblast differentiation. Non-treated group did not show mineralized nodule. CN-C or CN-H-treated group showed minerlaized nodules in 16 days. In northern blot analysis, CN-C or CN-H-treated group showed the elevated expression of ALP, BSP and OC in 16 days. These results suggest the possibility to develop deer antler as a bone regenerative agent in periodontal therapy by showing the stimulating activity of deer antler on differentiation of osteoblast.

• The Journal of the Korean Society for Microbiology
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• v.21 no.3
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• pp.311-329
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• 1986

The experimental exposure of animals to sources of ultraviolet radiation (UVR) which emit their energy primarily in the UVB region (280-320nm) is known to result in a number of well-described changes in the recipient's immune competence. Two such changes include a depressed capacity to effectively respond immunologically to transplants of syngeneic UVR tumors and a markedly reduced responsiveness to known inducers of delayedtype (DTH) and contact hypersensitivity (CH) reactions. The results of experiments that were designed to elucidate the mechanisms responsible for UVR-induced immunomodulation have implicated: 1) an altered pattern of lymphocyte recirculation, 2) suppressor T cells(Ts), 3) deviations in systemic antigen presenting cell (APC) potential. 4) changes in the production of interleukin-1-like molecules, and 5) the functional inactivation of epidermal Langerhans cells in this process. The exposure of skin to UVR, therefore, causes a number of both local and systemic alterations to the normal host immune system. In spite of this seeming complexity and diversity of responses, our recent studies have established that each of the UVR-mediated changes is probably of equal importance to creating the UVR-induced immunocompromised state. Normal animals were exposed to low dose UVR radiation on their dorsal surfaces under conditions where a $3.0\;cm^2$ area of skin was physically protected from the light energy. Contact sensitization of these animals with DNFB, to either the irradiated or protected back skin, resulted in markedly reduced CH responses. This was observed in spite of a normal responsiveness following the skin sensitization to ventral surfaces of the UVR-exposed animals. Systemic treatment of the low dose UVR recipients with the drug indomethacin (1-3 micrograms/day) during the UVR exposures resulted in a complete reversal of the depressions observed following DNFB sensitization to "protected" dorsal skin while the altered responsiveness found in the group exposed to the skin reactive chemical through directly UVR-exposed sites was maintained. These studies implicate the importance of EC as effective APC in the skin and also suggest that some of the systemic influences caused by UVR exposure involve the production of prostaglandins. This concept was further supported by finding that indomethacin treatment was also capable of totally reversing the systemic depressions in CH responsiveness caused by high dose UVR exposure (30K joules/$m^2$) of mice. Attempts to analyze the cellular mechanisms responsible established that the spleens of all animals which demonstrated altered CH responses, regardless of whether sensitization was through a normal or an irradiated skin site, contained suppressor cells. Interestingly, we also found normal levels of T effector cells in the peripheral lymph nodes of the UVR-exposed mice that were contact sensitized through normal skin. No effector cells were found when skin sensitization took place through irradiated skin sites. In spite of such an apparent paradox, insight into the probable mechanisms responsible for these observations was provided by establishing that UVR exposure of skin results in a striking and dose-dependent blockade of the efferent lymphatic vessels in all peripheral lymph nodes. Therefore, the afferent phases of immune responses can apparently take place normally in UVR exposed animals when antigen is applied to normal skin. The final effector responses, however, appear to be inhibited in the UVR-exposed animals by an apparent block of effector cell mobility. This contrasts with findings in the normal animals. Following contact sensitization, normal animals were also found to simultaneously contain both antigen specific suppressor T cells and lymph node effector cells. However, these normal animals were fully capable of mobilizing their effector cells into the systemic circulation, thereby allowing a localization of these cells to peripheral sites of antigen challenge. Our results suggest that UVR is probably not a significant inducer of suppressor T-cell activity to topically applied antigens. Rather, UVR exposure appears to modify the normal relationship which exists between effector and regulatory immune responses in vivo. It does so by either causing a direct reduction in the skin's APC function, a situation which results in an absence of effector cell generation to antigens applied to UVR-exposed skin sites, inhibiting the capacity of effector cells to gain access to skin sites of antigen challenge or by sequestering the lymphocytes with effector cell potential into the draining peripheral lymph nodes. Each of these situations result in a similar effect on the UVR-exposed host, that being a reduced capacity to elicit a CH response. We hypothesize that altered DTH responses, altered alloresponses, and altered graft-versus-host responses, all of which have been observed in UVR exposed animals, may result from similar mechanisms.

• Korean Journal of Plant Resources
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• v.31 no.3
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• pp.218-227
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• 2018

In this study, we investigated the effect of the extracts from Vaccinium oldhamii on cell proliferation and the regulatory mechanisms of cyclin D1 protein level in human cancer cells. The branch extracts from Vaccinium oldhamii (VOB) showed higher inhibitor effect against the cell growth than leave extracts (VOL) and fruit extracts (VOF) in human colorectal cancer, breast cancer, prostate cancer, non-small lung cancer, pancreatic cancer and liver cancer cells. In addition, VOB decreased cyclin D1 level at both protein and mRNA level. MG132 treatment attenuated VOB-mediated cyclin D1 downregulation. A point mutation of threonine-286 to alanine attenuated cyclin D1 degradation by VOB. In addition, the inhibition of nuclear export by leptomycin B (LMB) attenuated cyclin D1 degradation by VOB. But, the treatment of PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), LiCl ($GSK3{\beta}$ inhibitor), LY294002 (PI3K inhibitor) or BAY 11-7082 ($I{\kappa}K$ inhibitor) did not affect VOB-induced cyclin D1 degradation. In conclusion, VOB induced cyclin D1 degradation through redistribution of cyclin D1 from the nucleus to cytoplasm via T286 phosphorylation of cyclin D1, which resulted in the inhibition of cancer cell proliferation.

• Molecules and Cells
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• v.38 no.5
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• pp.402-408
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• 2015

Protein O-GlcNAcylation, dictated by cellular UDP-N-acetylglucosamine (UDP-GlcNAc) levels, plays a crucial role in posttranslational modifications. The enzyme GlcNAc kinase (NAGK, E.C. 2.7.1.59) catalyzes the formation of GlcNAc-6-phosphate, which is a major substrate for the biosynthesis of UDP-GlcNAc. Recent studies have revealed the expression of NAGK in different types of cells especially in neuronal dendrites. Here, by immunocytochemistry (ICC) and immunonucleochemistry (INC) of cultured rat hippocampal neurons, HEK293T and GT1-7 cells, we have showed that NAGK immuno-reactive punctae being present in the nucleoplasm colocalized with small nuclear ribonucleoprotein-associated protein N (snRNPN) and p54NRB, which are speckle and paraspeckle markers, respectively. Furthermore, NAGK IR cluster was also found to be colocalized with GTF2H5 (general transcription factor IIH, polypeptide 5) immuno reactive punctae. In addition, relative localization to the ring of nuclear lamin matrix and to GlcNAc, which is highly enriched in nuclear pore complexes, showed that NAGK surrounds the nucleus at the cytoplasmic face of the nuclear outer membrane. By in situ proximity ligation assay (PLA) we confirmed the colocalization of NAGK with snRNPN in the nucleus and in dendrites, while we also verified the interactions of NAGK with p54NRB, and with GTF2H5 in the nucleus. These associations between NAGK with speckle, paraspeckle and general transcription factor suggest its regulatory roles in gene expression.

• Journal of Food Hygiene and Safety
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• v.31 no.3
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• pp.141-152
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• 2016

The demand to develop more safe and efficient methods for treating allergic patient is now continuously growing due to the increasing prevalence of allergic diseases. Probiotics are endogenous microbial flora that gives health benefits within hosts. Probiotics are now considered as one of solutions to treat allergic patients since recent evidence shows that some of probiotics have immunomodulatory function. Also, the treatment of probiotics to patients is relatively safer than other anti-inflammatory agents. In this review, we summarized on immunomodulatory function of some probiotics which show preventive or therapeutic effects on major allergic diseases such as atopic dermatitis, allergic rhinitis, asthma, or food allergy. Based on previous literature, the treatment of probiotics can alleviate the symptoms of allergic diseases via balancing $Th_1/Th_2$ response or increasing the number of regulatory T ($T_{reg}$) cells.

• Journal of Periodontal and Implant Science
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• v.27 no.2
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• pp.425-441
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• 1997

The neuropeptide substance P(SP) has been implicated in the mediation of inflammation and immune-mediated disease such as arthritis. Recently, it was reported that SP was markedly increased around the blood vessels in inflamed gingiva as well as in close association with the inflammatory cell infiltrate. These results support that SP may contribute to the pathophysiology of neuronal inflammation in human periodontal tissues. SP may regulate inflammatory/immune responses by stimulating the proliferation of human T cells, differentiation and antibody-secreting potential of B cells, macrophage respiratory burst, connective tissue proliferation, and the secretion of cytokines from monocytes and T cells. Here, I studied potential role of SP as a costimulatory chemical signal in inflammatory/immune responses, by determining the released proinflammatory cytokines such as $MIP-1{\alpha}$, $IL-1{\beta}$, and IL-6 from culture supernatants of homogeneous immune cell lines. Serum free cell supernatants were concentrated with TCA precipitation, fractionated with SDS-PAGE, and subjected into western blot analysis. Among 15 cell lines tested, macrophage/monocyte cell line RAW264.7 and WRl9m.1 showed the highest level of induction of $MIP-1{\alpha}$ when stimulated with LPS. Discrete IL-6 bands with multiple forms of molecular mass were detected from supernatants of B cell lines A20(32kDa), Daudi(32, 35kDa), and SKW6.4(29kDa), which were expressed constitutively. $IL-1{\beta}$ could not be detected by the method of western blot analysis from supernatants of all cell lines tested except RAW264.7, WRl9m.1, and erythroid cell line K562 which showed the least amount of $IL-{\beta}$ secretion. SP $10^{-9}M$ with suboptimal dose of LPS treatment showed synergistic induction of $MIP-1{\alpha}$ release from RAW264.7 or WR19m.1, and also IL-6 release from A20, but this synergism is not the case in costimulation of RAW264.7 or WRl9m.1 with SP $10^{-9}M$ and TPA. Although treatment of T cell line CTLL-R8 with SP $10^{-7}M$ or PHA+TPA induced modest level of $MIP-1{\alpha}$ secretion, synergism was not observed when they are applied together. These findings all together suggest the possibility of a regulatory role of SP in inflammatory/immune reaction through differential modulation of bioactivities of other chemical cosignals.

• Laboraroty Animal Research
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• v.34 no.4
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• pp.302-310
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• 2018

CD47 (integrin-associated protein), a multi-spanning transmembrane protein expressed in all cells including red blood cells (RBCs) and leukocytes, interacts with signal regulatory protein ${\alpha}$ ($SIRP{\alpha}$) on macrophages and thereby inhibits phagocytosis of RBCs. Recently, we generated a novel C57BL/6J CD47 knockout ($CD47^{-/-}$ hereafter) mouse line by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease, and here report their hematological phenotypes. On monitoring their birth and development, $CD47^{-/-}$ mice were born viable with a natural male-to-female sex ratio and normally developed from birth through puberty to adulthood without noticeable changes in growth, food/water intake compared to their age and sex-matched wild-type littermates up to 26 weeks. Hematological analysis revealed a mild but significant reduction of RBC counts and hemoglobin in 16 week-old male $CD47^{-/-}$ mice which were aggravated at the age of 26 weeks with increased reticulocyte counts and mean corpuscular volume (MCV), suggesting hemolytic anemia. Interestingly, anemia in female $CD47^{-/-}$ mice became evident at 26 weeks, but splenomegaly was identified in both genders of $CD47^{-/-}$ mice from the age of 16 weeks, consistent with development of hemolytic anemia. Additionally, helper and cytotoxic T cell populations were considerably reduced in the spleen, but not in thymus, of $CD47^{-/-}$ mice, suggesting a crucial role of CD47 in proliferation of T cells. Collectively, these findings indicate that our $CD47^{-/-}$ mice have progressive hemolytic anemia and splenic depletion of mature T cell populations and therefore may be useful as an in vivo model to study the function of CD47.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.48 no.4
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• pp.331-342
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• 2022

Obesity is one of the metabolic diseases caused by excessive differentiation and accumulation of adipose tissue due to an imbalance between energy intake and consumption. In this study, we investigated the anti-obesity effect of SliMax, a natural-derived 6 compounds mixture, by using 3T3-L1 cells. As a result, SliMax showed the inhibitory effect on adipocyte differentiation through down-regulation of the PPARγ and C/EBPα expression, which are known to regulate the late adipogenesis stage. In the process of lipolysis on differentiated 3T3-L1 cells, SliMax accelerated decomposition of large-sized unilocular lipid droplet into numerous small-sized multilocular lipid droplets through up-regulation of the expression of lipolysis-related proteins ATGL and HSL. Finally, in order to confirm the effect of SliMax on induction of brown adipocyte, the expression of UCP-1 and the amount of mitochondria were confirmed by immunofluorescent staining, and as a result, SliMax increased the expression of UCP-1 and the amount of mitochondria in fat cells. Taken together, those results suggest that SliMax, a naturally-derived mixture, have a potential to be anti-obesity agent through exerting inhibitory effect on the formation of lipid droplet by suppression of adipogenesis and stimulation of lipolysis, and browning effect associated with generation of heat energy and energy consumption.