• Mycobiology
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• 제30권3호
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• pp.160-165
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• 2002

Aspergillus niger has been used as a host to express many heterologous proteins. It has been known that the presence of an- abundant protease is a limiting factor to express a heterologous protein. The protease deficient mutant of A. niger was obtained using UV-irradiation. A total of $1{\times}10^5$ spores were irradiated with $10{\sim}20%$ survival dose of UV, 600 $J/m^2$ at 280 nm, and the resulting spores were screened on the casein-gelatin plates. Ten putative protease deficient mutants showing the reduced halo area around colonies were further analyzed to differentiate the protease deficient mutant from other mutant types. Among ten putative mutants, seven mutants showed significant growth defect on nutrient rich medium and two mutants appeared to be the secretory mutants, which resulted in the impaired secretion of extracellular proteins including proteases. A mutant $pro^--20$ showed reduced halo zone without any notable changes in growth rate. In addition, the starchdegrading and glucose oxidase activities in the culture filtrate of $pro^--20$ mutant showed the similar range as that of the parental strain, which suggested that the $pro^--20$ mutant ought to be the protease deficient mutant rather than a secretory mutant. The reduced proteolytic activity of the $pro^--20$ was demonstrated using SDS-fibrin zymography gel. The reduced extracellular proteolysis was quantified by casein degradation assay and, comparing with the parental strain, less than 30% residual extracellular protease activity was detected in the culture filtrate of the $pro^--20$ mutant. The bio-activity of an exogenously supplemented hGM-CSF(human Granulocyte-Macrophage Colony Stimulating Factor) in the culture filtrate of $pro^--20$ mutant was detected until eight times more diluted preparations than that of the parental strain.

• 대한수의학회지
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• 제52권4호
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• pp.231-238
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• 2012

The expression profiles of inflammatory cytokines viz. interleukins (IL)-6, IL-8, IL-12, granulocyte macrophage-colony stimulating factor, interferon-${\gamma}$ and tumor necrosis factor-${\alpha}$ in response to subclinical mastitis in indigenous cattle breed Kankrej (n = 6), Gir (Bos indicus) (n = 12) and crossbred (Bos taurus${\times}$Bos indicus) (n = 7) were investigated using quantitative real time PCR. Significant correlation (p < 0.05) was observed between total bacterial load and somatic cell count (SCC) in all three breeds of cattle. All the cytokines were observed to be up-regulated compared to cows with healthy quarters, however, level of their expression varied among three breeds of cattle. In Kankrej most cytokines were found to be transcribed to higher levels than in other two breeds; the milk had higher load of bacteria but not so high SCC, implying that Kankrej has a higher inherent resistance against mastitis. The results of present study indicated that mammary glands of crossbred cattle are more sensitive to bacterial infection than indigenous breed of cattle as they elicit immune response at lower bacterial load and result into higher SCC. Research on identification of factors responsible for differentially expressed cytokines profiles and use of cytokines as immunomodulatory tools can pave way for formulating control strategies against bovine mastitis.

• 대한한의학회지
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• 제24권1호
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• pp.74-83
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• 2003

Background : Production of cytokines by bronchial epithelial cells may contribute to the local accumulation of inflammatory cells in patients with bronchial asthma. In many recent studies, molecular biological methods have been used to investigate the role of cytokines in pathogenesis and new therapeutic targets of asthma. Objective : We aimed to identify the dose-dependent inhibitory effects of Socheongryong-tang and Socheongryong-tang plus Sasam (Adenophorae Radix) on the mRNA expressions of Interleukin (IL)-6, IL-8 and granulocyte macrophage colony stimulating factor (GM-CSF) involved in the asthma model. Materials and Methods : In this study, BEAS-2B cell lines, human epithelial cells, were used. These cells were stimulated by tumor necrosis factor $(TNF)-{\alpha},{\;}IL-1{\beta}$ and histamine for artificial inflammatory expression. ${\beta}-actin$ messenger RNA (mRNA) was used for the internal standard. After each 24 hours of the Socheongryong-tang (小靑龍湯) and Socheongryong-tang plus Sasam (小靑 龍湯加沙蔘) treatment, total cellular RNAs were collected by applying RNAzol directly to the living cells. Then the transcriptional activities of IL-6, IL-8 and GM-CSF were measured by RT-PCR with electrophoresis. Results : In the Socheongryong-tang (小靑龍湯) study, the mRNA expressions of IL-6, IL-8 and GM-CSF were significantly inhibited compared to that of the control group (p<0.05). In the Socheongryong-tang plus Sasam (小靑龍湯加沙蔘) study, the mRNA expressions of IL-6, IL-8 and GM-CSF were significantly inhibited compared to that of the control group (p<0.05). Conclusions : This study shows that Socheongryong-tang (小靑龍湯) and Socheongryong-tang plus Sasam (小靑龍湯加沙蔘) have dose-dependent inhibitory effects on the mRNA expressions of IL-6, IL-8 and GM-CSF in human epithelial cells, so these herbal medicines may inhibit the inflammatory process of asthma. Advanced studies are required to investigate the mechanisms of inhibition by herbal medicine in the asthma model.

• 대한암한의학회지
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• 제25권1호
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• pp.11-24
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• 2020

Objective : Radiodermatitis is a common sequelae in 95% of patients receiving radiation therapy, which is important to be well managed as it can affect the patient's quality of life as well as the cancer treatment schedule. The aim of this study is to review and summarize the interventions available for the treatment of acute radiodermatitis, including traditional Korean medicine, and to propose treatment algorithms for clinicians. Methods : To collect studies about managements for radiodermatitis, domestic and foreign database were used such as Korean journal of traditional knowledge portal (KTKP), Korean studies information service system (KISS), national discovery or science leaders (NDSL), and oriental medicine advance searching integrated system (OASIS), Pubmed, Google scholar and Scopus. Results : Thirty-two studies were selected. There were nine studies on usual care and dressing, eleven studies on chemical agents, two studies on biological preparations, and ten studies on herbal medicines. Conclusion : Hygienic options and dressings have proved to be useful in the management of radiodermatitis. Chemical agents such as corticosteroid, statin, and topical antibiotic agent have proved to alleviate symptoms and severity, regenerate damaged skin, and prevent secondary infection. In biological preparations, EGF (epidermal growth factor) and GM-CSF (granulocyte-macrophage colony-stimulating factor) could be used to protect skin and prevent radiodermatitis. For herbal medicines, Calendula, catechin, β-sitosterol, and Jaungo (紫雲膏) may be effective for symptoms including pain, itching, and burning sensation induced by radiation therapy. Because of some research with conflicting results, further studies are needed to propose an algorithm for more optimal treatments.

• Advances in Traditional Medicine
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• 제5권3호
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• pp.188-194
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• 2005

The Angelicae dahuricae radix (ADR) has been used a traditional medicine to treat acne, erythema, headache, toothache, sinusitis, colds, and flu in Korea, Japan and China. Here, we report the effect of ADR on compound 48/80-induced ear-swelling and the phorbol myristate acetate (PMA) plus calcium ionophore A23187-induced inflammatory cytokine secretion in the human mast cell line, HMC-1. ADR dose-dependently inhibited the ear-swelling response induced by intradermal injection of compound 48/80, In vitro model, PMA plus A23187 significantly increased interleukin $(IL)-1{\beta}$, IL-8, granulocyte macrophage colony stimulating factor (GM-CSF), and tumor necrosis factor $(TNF)-{\alpha}$ secretion compared with media control. We also show that the increased cytokines $IL-1{\beta}$, IL-8, GM-CSF, and $TNF-{\alpha}$ level was significantly inhibited by treatment of ADR. In addition, ADR partially blocked PMA plus A23187-induced extracelluar signal-regulated kinases phosphorylation. These results suggest that ADR might explain its beneficial effect in the treatment of mast cell-mediated inflammatory diseases.

• Journal of Ginseng Research
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• 제39권4호
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• pp.322-330
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• 2015

Background: UV-irradiated keratinocytes secrete various proinflammatory cytokines. UV-induced skin damage is mediated by growth factors and proinflammatory cytokines such as granulocyte macrophage colony stimulating factor (GM-CSF). In a previous study, we found that the saponin of Korean Red Ginseng (SKRG) decreased the expression of GM-CSF in UVB-irradiated SP-1 keratinocytes. In this study, we attempted to find the inhibitory mechanism of SKRG on UVB-induced GM-CSF expression in SP-1 keratinocytes. Methods: We investigated the inhibitory mechanism of SKRG and ginsenosides from Panax ginseng on UVB-induced GM-CSF expression in SP-1 keratinocytes. Results: Treatment with SKRG decreased the expression of GM-CSF mRNA and protein induced by irradiation of UVB in SP-1 keratinocytes. The phosphorylation of ERK was induced by UVB at 10 min, and decreased with SKRG treatment in SP-1 keratinocytes. In addition, treatment with SKRG inhibited the UVB-induced phosphorylation of epidermal growth factor receptor (EGFR), which is known to be an upstream signal of ERK. From these results, we found that the inhibition of GM-CSF expression by SKRG was derived from the decreased phosphorylation of EGFR. To identify the specific compound composing SKRG, we tested fifteen kinds of ginsenosides. Among these compounds, ginsenoside-Rh3 decreased the expression of GM-CSF protein and mRNA in SP-1 keratinocytes. Conclusion: Taken together, we found that treatment with SKRG decreased the phosphorylation of EGFR and ERK in UVB-irradiated SP-1 keratinocytes and subsequently inhibited the expression of GM-CSF. Furthermore, we identified ginsenoside-Rh3 as the active saponin in Korean Red Ginseng.

• 생명과학회지
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• 제19권12호
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• pp.1690-1697
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• 2009

CD11c와 CD80 및 CD86과 같은 보조 수용체는 주로 수지상 세포에서 발현되는 세포 표지 인자이다. 본 연구에서는 KG-1, HL-60, NB4 및 THP-1 세포와 같은 여러 종류의 백혈병 세포를 이용하여 이들 세포에 재조합 Flt-3 리간드를 처리하였을 때 수지상 세포의 표면 인자인 CD11c의 발현에 어떠한 변화가 있는가를 조사하였다. KG-1 세포뿐만 아니라 NB4세포와 HL-60 세포에서도 Flt-3 수용체가 발현됨을 확인하였으나 THP-1 세포에서는 이들 수용체가 발현되지 않았다. KG-1 세포를 Flt-3 리간드나 granulocyte macrophage-colony stimulating factor (GM-CSF)와 tumor necrosis factor (TNF)-$\alpha$를 섞은 배양액에서 배양하였을 때 세포 증식은 억제되었으며 CD11c 발현은 현저히 증가되었다. 그러나 Flt-3 리간드를 처리한 KG-1세포에서는 GM-CSF와 TNF-$\alpha$를 처리한 세포에서와는 다르게 major histocompatibility complex (MHC)-I 및 MHC-II의 발현은 증가되지 않았다. Flt-3 리간드는 HL-60 세포와 NB4 세포의 CD11c 발현도 증가시켰으나 THP-1 세포에서는 아무런 영향이 없었다. CD11c의 발현과 비교하여 CD11b의 발현은 Flt-3 리간드에 의하여 KG-1 세포에서는 약하게 증가하였으나 NB4 세포와 HL-60 세포에서는 증가되지 않았다. KG-1 세포를 Flt-3 리간드로 처리하였을 때 extracellular signal-regulated kinase-1/2 (ERK-1/2)와 p38-mitogen-activated protein kinase (p38-MAPK)의 단백질 인산화가 증가되었으며 Flt-3 리간드에 의한 CD11c 발현의 증가는 MEK의 억제제인PD98059에 의하여 사라짐을 확인하였다. 본 연구 결과는 Flt-3 수용체 리간드의 처리에 의하여 $CD34^+$ myelomonocyte분화 단계인 KG-1 세포와 promyelocyte 분화 단계의 백혈병 세포에서 수지상 세포와 유사한 세포 형으로 분화된다는 것을 보였고 Flt-3 수용체 리간드에 의한 이들 백혈병 세포의 수지상 세포유사 세포로의 분화는 ERK-1/2의 활성화에 의하여 일어날 수 있음을 보여 준다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.320-323
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• 2003

본 연구에서는 형질전환된 식물세포배양으로 생산한 (hGM-CSF)를 cationic exchange resin인 CM-sepharose와 anionic exchange resin인 DEAE-sepharose에 통과키며 pH의 변화를 주었을 때 흡착 여부를 확인하였다. hGM-CSF는 pH가 5-7사이의 범위에서 가장 안정함을 보였으며 이 결과를 바탕으로 각각의 pH 범위를 결정하였다. Buffer exchange를 했을 때 cationic exchange resin인 CM-sepharose의 경우 pH 4.8에서 상대적으로 가장 높은 흡착율(77%)을 보였으며 anionic exchange resin인 DEAE-sepharose를 이용한 흡착에서는 pH 5.5에서 높은 흡착율(74%)을 보였다. 이러한 결과를 바탕으로 buffer exchange 없이 hGM-CSF가 secretion된 배지를 pH를 맞춘 후 좁은 범위에서의 흡착 실험을 수행하였다. 그 결과 pH 4.6에서 CM-sepharose를 이용했을 때 흡착율이 84%로 가장 좋았다. 이러한 결과를 이용하면 외래 단백질을 생산하는 식물세포 배양시에 가장 문제가 되는 protease에 의한 목적 단백질의 degradation을 해결할 수 있는 in situ adsorption이 가능하리라 사료된다.

• IMMUNE NETWORK
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• 제5권2호
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• pp.110-116
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• 2005

Background: As an attempt to develop a strategy to improve the protective immune response to GM-CSF-secreting CT26 (GM-CSF/CT26) tumor vaccine, we have investigated whether the apoptogenic treatment of GM-CSF/CT26 prior to vaccination enhances the induction of anti-tumor immune response in mouse model. Methods: A carcinogeninduced mouse colorectal tumor, CT26 was transfected with GM-CSF gene using a retroviral vector to generate GM-CSF-secreting CT26 (CT26/GM-CSF). The CT26/GM-CSF was treated with ${\gamma}$-irradiation or mitomycin C to induce apoptosis and vaccinated into BALB/c mice. After 7 days, the mice were injected with a lethal dose of challenge live CT26 cells to examine the protective effect of tumor vaccination in vivo. Results: Although both apoptotic and necrotic CT26/GM-CSF vaccines were able to enhance anti-tumor immune response, apoptotic CT26/GM-CSF induced by pretreatment with ${\gamma}$-irradiation (50,000 rads) was the most potent in generating the anti-tumor immunity, and thus 100% of mice vaccinated with the apoptotic cells remained tumor free for more than 60 days after tumor challenge. Conclusion: Apoptogenic pretreatment of GM-CSF-secreting CT26 tumor vaccine by ${\gamma}$-irradiation (50,000 rads) resulted in a significant enhancement in inducing the protective anti-tumor immunity. A rapid induction of apoptosis of CT26/GM-CSF tumor vaccine at the vaccine site might be critical for the enhancement in anti-tumor immune response to tumor vaccine.

• KSBB Journal
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• 제30권2호
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• pp.82-90
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• 2015

Plant cell immobilization can protect plant cells from shear forces and increase the stability of gene. An additional advantage of immobilization is the easiness for performing continuous culture with cell recycling. Therefore plant cell immobilization can overcome the limitations of plant cell applications. In addition, target protein should be selected from pharmaceutical proteins to get rid of low expression level problem. The enhanced production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was investigated in immobilized Nicotiana tabacum suspension cell cultures. When the cells were immobilized in polyurethane foam, specific production of hGM-CSF was higher than that in alginate bead immobilization. Optimum continuous culture condition was the addition of 60 g/L sucrose in growth media with exchanging media every 6 day. Under the same condition, specific hGM-CSF production was 7 times higher in a 500-mL spinner flask than that in 100-mL Erlenmeyer flasks. Therefore, development of an effective immobilization process would be possible when the advantage of easy cell recycling was used. Consequently, enhanced production of target proteins could be possible in immobilized continuous cultures when the advantages of immobilization were applied.