• Journal of Microbiology and Biotechnology
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• 제32권4호
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• pp.484-492
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• 2022

Lichenase is an enzyme mainly implicated in the degradation of polysaccharides in the cell walls of grains. Emerging evidence shows that a highly efficient expression of a thermostable recombinant lichenase holds considerable promise for application in the beer-brewing and animal feed industries. Herein, we cloned a lichenase gene (CelA203) from Bacillus subtilis B110 and expressed it in E. coli. This gene contains an ORF of 729 bp, encoding a protein with 242 amino acids and a calculated molecular mass of 27.3 kDa. According to the zymogram results, purified CelA203 existed in two forms, a monomer, and a tetramer, but only the tetramer had potent enzymatic activity. CelA203 remained stable over a broad pH and temperature range and retained 40% activity at 70℃ for 1 h. The K_m and V_max of CelA203 towards barley β-glucan and lichenan were 3.98 mg/ml, 1017.17 U/mg, and 2.78 mg/ml, 198.24 U/mg, respectively. Furthermore, trisaccharide and tetrasaccharide were the main products obtained from CelA203-mediated hydrolysis of deactivated oat bran. These findings demonstrate a promising role for CelA203 in the production of oligosaccharides in animal feed and brewing industries.

• Parasites, Hosts and Diseases
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• 제60권2호
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• pp.117-126
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• 2022

Cystatin, a cysteine protease inhibitor found in many parasites, plays important roles in immune evasion. This study analyzed the molecular characteristics of a cystatin from Fasciola hepatica (FhCystatin) and expressed recombinant FhCystatin (rFhcystatin) to investigate the immune modulatory effects on lipopolysaccharide-induced proliferation, migration, cytokine secretion, nitric oxide (NO) production, and apoptosis in mouse macrophages. The FhCystatin gene encoded 116 amino acids and contained a conserved cystatin-like domain. rFhCystatin significantly inhibited the activity of cathepsin B. rFhCystatin bound to the surface of mouse RAW264.7 cells, significantly inhibited cell proliferation and promoted apoptosis. Moreover, rFhCystatin inhibited the expression of cellular nitric oxide, interleukin-6, and tumor necrosis factor-α, and promoted the expression of transforming growth factor-β and interleukin-10. These results showed that FhCystatin played an important role in regulating the activity of mouse macrophages. Our findings provide new insights into mechanisms underlying the immune evasion and contribute to the exploration of potential targets for the development of new drug to control F. hepatica infection.

• IMMUNE NETWORK
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• 제23권2호
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• pp.14.1-14.14
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• 2023

Immune status including the immune cells and cytokine profiles has been implicated in the development of endometriosis. In this study, we analyzed Th17 cells and IL-17A in peritoneal fluid (PF) and endometrial tissues of patients with (n=10) and without (n=26) endometriosis. Our study has shown increased Th17 cell population and IL-17A level in PF with endometriosis patients. To determine the roles of IL-17A and Th17 cells in the development of endometriosis, the effect of IL-17A, major cytokine of Th17, on endometrial cells isolated from endometriotic tissues was examined. Recombinant IL-17A promoted survival of endometrial cells accompanied by increased expression of anti-apoptotic genes, including Bcl-2 and MCL1, and the activation of ERK1/2 signaling. In addition, treatment of IL-17A to endometrial cells inhibited NK cell mediated cytotoxicity and induced HLA-G expression on endometrial cells. IL-17A also promoted migration of endometrial cells. Our data suggest that Th17 cells and IL-17A play critical roles in the development of endometriosis by promoting endometrial cell survival and conferring a resistance to NK cell cytotoxicity through the activation of ERK1/2 signaling. Targeting IL-17A has potential as a new strategy for the treatment of endometriosis.

• International Journal of Stem Cells
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• 제16권3호
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• pp.315-325
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• 2023

Background and Objectives: Glioblastoma (GBM) is an aggressive primary brain tumor characterized by its heterogeneity and high recurrence and lethality rates. Glioblastoma stem cells (GSCs) play a crucial role in therapy resistance and tumor recurrence. Therefore, targeting GSCs is a key objective in developing effective treatments for GBM. The role of Parathyroid hormone-related peptide (PTHrP) in GBM and its impact on GSCs remains unclear. This study aimed to investigate the effect of PTHrP on GSCs and its potential as a therapeutic target for GBM. Methods and Results: Using the Cancer Genome Atlas (TCGA) database, we found higher expression of PTHrP in GBM, which correlated inversely with survival. GSCs were established from three human GBM samples obtained after surgical resection. Exposure to recombinant human PTHrP protein (rPTHrP) at different concentrations significantly enhanced GSCs viability. Knockdown of PTHrP using target-specific siRNA (siPTHrP) inhibited tumorsphere formation and reduced the number of BrdU-positive cells. In an orthotopic xenograft mouse model, suppression of PTHrP expression led to significant inhibition of tumor growth. The addition of rPTHrP in the growth medium counteracted the antiproliferative effect of siPTHrP. Further investigation revealed that PTHrP increased cAMP concentration and activated the PKA signaling pathway. Treatment with forskolin, an adenylyl cyclase activator, nullified the antiproliferative effect of siPTHrP. Conclusions: Our findings demonstrate that PTHrP promotes the proliferation of patient-derived GSCs by activating the cAMP/PKA signaling pathway. These results uncover a novel role for PTHrP and suggest its potential as a therapeutic target for GBM treatment.

• 생명과학회지
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• 제24권1호
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• pp.86-91
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• 2014

Trichoplusia ni (T. ni) 세포는 사람 당 수송체를 이성질적으로 많은 양 생산하려 할 때 유용하게 사용되는, baculovirus 발현 시스템의 숙주세포로서 이용된다. 그러나 T. ni 세포에 존재하는 내재된 당 수송체의 높은 활동은, 발현된 외재적 당 수송체의 수송활성과 같은 직접적 증거 제시에 장애가 된다. 뿐만 아니라 곤충세포에 내재하는 당 수송체계의 특성에 대해서는 밝혀진 바가 거의 없다. 그래서 본 연구에서는 baculovirus 발현 시스템을 보다 잘 활용하기 위해 T. ni 세포의 2dGlc기질 수송에 D-fructose가 미치는 영향을 살펴 보았으며, T. ni 세포에 발현된 사람 당 수송체의 생물학적 활성을 보다 용이하게 검증하기 위해 발현된 수송체를 [$^3H$] cytochalasin B를 이용하여 photolabelling 하였다. 우선 감염되지 않은 세포와 recombinant AcMPV-GTL 감염시킨 T. ni 세포의 2dGlc uptake를 300 mM D-fructose가 있을 때와 없을 때, 그리고 $20{\mu}M$ cytochalasin B가 있을 때와 없을 때의 상황에서 살펴보았다. 감염되지 않은 세포에서의 육탄당 uptake는 D-fructose에 의해 강력하게 억제 되었으나 cytochalasin B에 의해서는 단지 미미한 억제 효과만을 보여주었다. 흥미롭게도 AcMPV-GTL 바이러스 감염된 T. ni 세포에서는 비록 2dGlc uptake율은 감염되지 않은 세포와 비교해 다소 낮았지만 육탄당 수송 억제 반응은 근본적으로 동일함을 보여 주었다. 또한 [$^3H$] cytochalasin B를 이용한 발현단백질 photolabelling에서는, L-glucose가 존재하는 상황 하에만 하나의 날카롭게 표지된 peak가, 바이러스 감염된 세포에서 관찰되었다. 감염되지 않은 세포에서는 이러한 peak는 관찰되지 않았다. 게다가 D-glucose 존재 하에서는 발현된 단백질의 photolabelling이 완전히 억제되어짐을 보여주어, labelling의 입체선택성(stereoselectivity)을 입증하였다.

• Parasites, Hosts and Diseases
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• 제54권1호
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• pp.21-29
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• 2016

The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of $Na^+$ and $H^+$ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of $Ca^{2+}$. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.

• 미생물학회지
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• 제51권3호
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• pp.271-279
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• 2015

락토페린(LF)는 철이온과 결합하는 당 단백질로서 항균, 항바이러스, 항진균 등의 기능을 가지고 있으며, 생체의 각종 체액으로부터 분비되는 다기능성 단백질이다. 본 연구에서는 사람의 락토페린(hLF)으로부터 유래된 N-lobe의 유전자를 분리하고 산업용 균주로서 많이 사용되는 메탄올자화 효모인 Pichia pastoris에서 발현시켰다. 재조합 사람 락토페린 N-lobe (rhLF-N)는 배양액으로 분비 발현되었으며, 3L 발효조에서 약 $458{\mu}g/ml$이 수준으로 생성되었다. rhLF-N을 정제한 다음 SDS-PAGE와 western blot으로 분석하여 분자량 35 kDa 단백질을 확인하였으며, hLF에 대한 항체를 이용하여 면역확산법으로 면역성을 확인하였다. rhLF-N의 mRNA 발현양상을 qRT-PCR로 분석한 결과 메탄올 첨가에 의한 발현 유도 후 2-3일째에 발현율이 가장 높았으며, 4일째에는 점차적으로 감소하였다. 정제한 rhLF-N을 이용하여 항균활성을 조사한 결과 Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, Burkholderia cepacia, Salmonella typhimurium과 같은 병원성 균에 대해 광범위한 항균활성을 보였으나, LF유래 항균 peptide들과 항균활성을 비교하였을 때, 항균력이 상대적으로 매우 떨어지는 것으로 나타났다. 비록 본 연구에서 발현한 rhLF-N은 항균력은 떨어지나, hLF에 비해 그 크기가 작고 배양조건 연구로 P. pastoris에서 대량 생산이 가능하며, 배양액으로 분비시킬 수 있기 때문에 정제 비용 등을 고려 할 때 산업적 응용에는 보다 유리할 것으로 사료된다.

• Journal of Periodontal and Implant Science
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• 제41권6호
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• pp.293-301
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• 2011

Purpose: We have previously reported that tetra-cell adhesion molecule (T-CAM) markedly enhanced the differentiation of osteoblast-like cells grown on anorganic bone mineral (ABM). T-CAM comprises recombinant peptides containing the Arg- Gly-Asp (RGD) sequence in the tenth type III domain, Pro-His-Ser-Arg-Asn (PHSRN) sequence in the ninth type III domain of fibronectin (FN), and the Glu-Pro-Asp-Ilu-Met (EPDIM) and Tyr-His (YH) sequence in the fourth fas-1 domain of ${\beta}$ig-h3. Therefore, the purpose of this study was to evaluate the cellular activity of osteoblast-like cells and the new bone formation on ABM coated with T-CAM, while comparing the results with those of synthetic cell binding peptide (PepGen P-15). Methods: To analyze the cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed, andto analyze gene expression, northernblot was performed. Mineral nodule formations were evaluated using alizarin red stain. The new bone formations of each group were evaluated using histologic observation and histomorphometrc analysis. Results: Expression of alkaline phosphatase mRNA was similar in all groups on days 10 and 20. The highest expression of osteopontin mRNA was observed in the group cultured with ABM/P-15, followed by those with ABM/T-CAM and ABM on days 20 and 30. Little difference was seen in the level of expression of collagen type I mRNA on the ABM, ABM/T-CAM, and ABM/P-15 cultured on day 20. There were similar growth and proliferation patterns for the ABM/T-CAM and ABM/P-15. The halo of red stain consistent with $Ca^{2+}$ deposition was wider and denser around ABM/T-CAM and ABM/P-15 particles than around the ABM particles. The ABM/T-CAM group seemed to have bone forming bioactivity similar to that of ABM/P-15. A complete bony bridge was seen in two thirds of the defects in the ABM/T-CAM and ABM/P-15 groups. Conclusions: ABM/T-CAM, which seemed to have bone forming bioactivity similar to ABM/P-15, was considered to serve as effective tissue-engineered bone graft material.

• 생명과학회지
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• 제22권4호
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• pp.552-558
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• 2012

사스(Severe acute respiratory syndrome, SARS)는 사람의 신종 폐렴인 중증 급성 호흡기 질환으로 신종 코로나바이러스, SARS-CoV에 의해 유발된다. 3CL protease는 SARS-CoV의 복제, 전사 및 단백질 합성을 조절하는 복제효소 복합단백질의 프로세싱에 결정적인 역할을 담당하는 중요한 효소이다. 따라서, 이 효소를 저해함으로써 SARS-CoV의 증식을 억제하고 사스의 증폭 및 확산을 막을 수 있다. SARS-3CL protease의 활성 저해물질의 탐색은 사스의 치료제 개발에 중요한 목표 중의 하나로 인식되고 있으며 이를 위해서는 활성형 SARS-3CL protease의 대량 생산이 필요하다. 본 연구에서는 활성형 SARS-3CL protease를 대량 생산하기 위하여 여러 가지 발현 벡터 및 단백질 발현 방법 등을 검토하였다. 그 결과, pET29a/3CLP 발현 벡터를 이용한 무세포 단백질 합성법이 SARS-3CL protease 생산에 최적 조건인 것으로 확인되었다. 또한 발현된 효소를 완전히 정제하여 그 특성을 분석한 결과, 본 효소는 무세포 단백질 합성계에서 전구체로 합성됨과 동시에 자가분해됨으로써 모든 단백질이 활성형인 성숙체 단백질로 전환되어 간단히 활성형 SARS-3CL protease 효소를 생산할 수 있음을 확인하였다.

• Journal of Microbiology and Biotechnology
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• 제16권12호
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• pp.1919-1927
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• 2006

Tumor cells genetically engineered to secrete cytokines are effective in tumor therapy, but various unexpected side effects are observed, which may result from the bulk activation of various bystander cells. In this study, we tested tumor vaccines expressing various membrane-bound forms of IL-2 (mbIL-2) on MethA fibrosarcoma cells to focus antitumor immune responses to CTL. Chimeric forms of IL-2 with whole CD4, deletion forms of CD4, and TNF were expressed on the tumor cell surface, respectively. Tumor clones expressing mbIL-2 or secretory form of IL-2 were able to support the cell growth of CTLL-2, an IL-2-dependent T cell line, and the proliferation of spleen cells from 2C TCR transgenic mice that are responsive to the $p2Ca/L^d$ MHC class I complex. Expression of mbIL-2 on tumor cells reduced the tumorigenicity of tumor cells, and the mice that once rejected the live IL-2/TNF tumor clone acquired systemic immunity against wild-type MethA cells. The IL-2/TNF clone was inferior to other clones in tumor formation, and superior in the stimulation of the CD8+ T cell population in vitro. These results suggest that the IL-2/TNF clone is the best tumor vaccine, and may stimulate CD8+ T cells by direct priming. Expression of IL-2/TNF on tumor cells may serve as an effective gene therapy method to ameliorate the side effects encountered in the recombinant cytokine therapy and the conventional cytokine gene therapy using the secretory form of IL-2.