• Asian-Australasian Journal of Animal Sciences
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• 제25권5호
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• pp.621-628
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• 2012

The FAT-1 protein is an n-3 fatty acid desaturase, which can recognize a range of 18- and 20-carbon n-6 substrates and transform n-6 polyunsaturated fatty acids (PUFAs) into n-3 PUFAs while n-3 PUFAs have beneficial effect on human health. Fat1 gene is the coding sequence from Caenorhabditis elegans which might play an important role on lipometabolism. To reveal the function of fat1 gene in bovine fetal fibroblast cells and gain the best cell nuclear donor for transgenic bovines, the codon of fat1 sequence was optimized based on the codon usage frequency preference of bovine muscle protein, and directionally cloned into the eukaryotic expression vector pEF-GFP. After identifying by restrictive enzyme digests with AatII/XbaI and sequencing, the fusion plasmid pEF-GFP-fat1 was identified successfully. The pEF-GFP-fat1 vector was transfected into bovine fetal fibroblast cells mediated by Lipofectamine2000$^{TM}$. The positive bovine fetal fibroblast cells were selected by G418 and detected by RT-PCR. The results showed that a 1,234 bp transcription was amplified by reverse transcription PCR and the positive transgenic fat1 cell line was successfully established. Then the expression level of fat1 gene in positive cells was detected using quantitative PCR, and the catalysis efficiency was detected by gas chromatography. The results demonstrated that the catalysis efficiency of fat1 was significantly high, which can improve the total PUFAs rich in EPA, DHA and DPA. Construction and expression of pEF-GFP-fat1 vector should be helpful for further understanding the mechanism of regulation of fat1 in vitro. It could also be the first step in the production of fat1 transgenic cattle.

• 한국미생물·생명공학회지
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• 제14권6호
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• pp.479-485
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• 1986

Bacillus amyloliquefaciens가 생성하는 액화형 $\alpha$-amylase의 구조유전자를 클로닝하기 위하여 이 균주 염색체의 2 - 5kb 획분을 분리하여 Mbo I으로 부분분해한 후 pUB110플라스미드의 BamHI 부위에 삽입하여 hybrid vector pSKS3 를 제작하였다. 이 재조합플라스미드를 세한수식결손 세포인 Bacillus subtilis ED 107에서 subcloning한 후 당화형 $\alpha$-amylase를 생성하는 Bacillus subtilis NA 64의 $\alpha$-amylase결실변이주 Bacills subtilis KM 107 주에서 형질 발현시켰다. 이때 형질전환빈도는 $10^{-5}$ - $10^{-7}$정도였으며 그중 약 50%가 $\alpha$-amylase 분비능이 있었으나 거의 대부분이 쉽게 실활되었다. 최종선별과정에서 $\alpha$-amylase를 가장 강하게 생성하는 형질전환주 Bacillus subtilis KM213으로부터 재 추출한 재조합플라스미드 pSKS3는 5.7kb 삽입된 단편이 BamH I/Mbo I 부위에 결합되어 있었다. pSKS3에 클로닝된 유전자에 의해 발현되는 $\alpha$-amylase 활성은 B. amyloliquefaciens 활성의 약 25%였으며, 이 pSKS3에 의해 발현되는 $\alpha$-amylase 단백은 B.amyloliquefaciens의 $\alpha$-amylase와 동일한 전기영동성을 나타내었음을 볼 때 pSKS3에 클로닝된 유전자는 B. amyloliquefaciens에서 유래함을 알 수 있었다.

• The Korean Journal of Physiology and Pharmacology
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• 제4권5호
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• pp.369-378
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• 2000

This study was undertaken to investigate an involvement of nitroxergic innervation in gastric smooth muscle of rat. Isometric tension study, the measurement of single cell length, NADPH diaphorase stain of smooth muscle layers and neuronal nitric oxide synthase (nNOS) western blotting were performed. Sodium nitroprusside (SNP), a nitric oxide donor, relaxed the muscle strips precontracted by acetylcholine (ACh) in a concentration-dependent manner. Pretreatment of L-arginine decreased the contraction induced by electric field stimulation (EFS). Pretreatment of $N^G-nitro-L-arginine$ methyl ester (L-NAME), a NOS inhibitor, increased the EFS-induced contractions. LY 83583, a guanylate cyclase (GC) inhibitor, reversed the inhibitory actions of L-arginine on the muscle contractions. The effects of L-Arginine, L-NAME and LY 83583 on ACh-induced contractions were not significant. L-arginine reduced the EFS-induced contraction in circular muscle, whereas L-NAME enhanced the EFS-induced contraction in longitudinal strips. By EFS, the phasic contractions appeared approximately $20{\sim}25$ seconds later. L-NAME significantly shortened the delay time to about $2{\sim}3$ seconds. In single cell study, ACh contracted gastric smooth muscle cells, SNP relaxed the cells, and the latter also inhibited the ACh-induced contraction. LY 83583 enhanced the ACh-induced contraction and antagonized SNP-induced relaxation. NADPH diaphorase activity was assessed by a histochemistry, nitroblue tetrazolium (NTB) staining. Positive staining was observed in both circular and longitudinal muscle layers. L-arginine increased the staining, while L-NAME decreased the staining. Western blotting for nNOS proved the presence of nNOS in rat gastric smooth muscle. EFS and additional $Ca^{2+}$ increased nNOS protein expression. These results suggest that in rat stomach, both circular and longitudinal muscle layers are innervated with nitroxergic nerves which relax the gastric smooth muscle via NO-cGMP pathway.

• The Korean Journal of Physiology and Pharmacology
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• 제18권1호
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• pp.25-32
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• 2014

Nitric oxide (NO) is recognized as a mediator and regulator of inflammatory responses. NO is produced by nitric oxide synthase (NOS), and NOS is abundantly expressed in the human dental pulp cells (HDPCs). NO produced by NOS can be cytotoxic at higher concentrations to HDPCs. However, the mechanism by which this cytotoxic pathway is activated in cells exposed to NO is not known. The purpose of this study was to elucidate the NO-induced cytotoxic mechanism in HDPCs. Sodium nitroprusside (SNP), a NO donor, reduced the viability of HDPCs in a dose- and time-dependent manner. We investigated the in vitro effects of nitric oxide on apoptosis of cultured HDPCs. Cells showed typical apoptotic morphology after exposure to SNP. Besides, the number of Annexin V positive cells was increased among the SNP-treated HDPCs. SNP enhanced the production of reactive oxygen species (ROS), and N-acetylcysteine (NAC) ameliorated the decrement of cell viability induced by SNP. However, a soluble guanylate cyclase inhibitor (ODQ) did not inhibited the decrement of cell viability induced by SNP. SNP increased cytochrome c release from the mitochondria to the cytosol and the ratio of Bax/Bcl-2 expression levels. Moreover, SNP-treated HDPCs elevated activities of caspase-3 and caspase-9. While pretreatment with inhibitors of caspase (z-VAD-fmk, z-DEVD-fmk) reversed the NO-induced apoptosis of HDPCs. From these results, it can be suggested that NO induces apoptosis of HDPCs through the mitochondria-dependent pathway mediated by ROS and Bcl-2 family, but not by the cyclic GMP pathway.

• 마이크로전자및패키징학회지
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• 제17권2호
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• pp.63-67
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• 2010

본 연구에서는 P3HT와 PCBM 물질을 전자도너와 억셉터 광활성층 물질로 사용하여 벌크이종접합 구조를 갖는 Glass/ITO/PEDOT:PSS/P3HT-PCBM/Al 구조의 유기박막태양전지를 제작하였다. P3HT와 PCBM은 각각 0.5 wt%의 농도로 톨루엔 용액에 용해하였다. 광활성층 농도를 최적화하기 위하여 P3HT:PCBM= 3:4, 4:4, 4:3 wt%의 농도비로 소자를 제작하고, 농도비에 따른 전기적 특성을 조사하였다. 또한 활성층의 후속열처리 온도가 소자의 전기적 특성에 미치는 영향을 조사하였다. P3HT와 PCBM의 농도비가 4:4 wt%의 비율에서 가장 우수한 전기적 특성을 나타내었으며, 이때 단락전류밀도 ($J_{SC}$), 개방전압 ($V_{OC}$), 및 충실인자 (FF)는 4.7 $mA/cm^2$, 0.48 V 및 43.1%를 각각 나타내었다. 또한 전력변환효율(PCE)은 0.97%의 값을 얻었다. 최적화된 농도비를 갖는 태양전지 소자에 대해 $150^{\circ}C$에서 5분, 10분, 15분, 20분간 후속 열처리를 실시한 결과 P3HT 전자도너의 흡광계수가 증가하는 경향을 보였다. 후속 열처리 조건이 $150^{\circ}C$에서 15분인 경우 전기적 특성이 열처리 하지 않은 소자에 비해 특성이 개선되었다. 즉, 이때의 전기적 특성은 $J_{SC},\;V_{OC}$, FF, PCE의 값이 각각 7.8 $mA/cm^2$, 0.55 V, 47%, 2.0%를 나타내었다.

• Applied Microscopy
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• 제37권4호
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• pp.249-258
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• 2007

이자섬은 이자를 구성하는 외분비조직에 둘러싸여 존재하는 내분비세포의 집단으로, 이자섬에서 분비되는 인슐린은 $\beta$세포에서 분비되는 호르몬이며, 세포질의 리보좀에서 합성되고 골지체를 경유하여 세포질로 방출되는 기작을 가지고 있다. 충분한 양의 이자섬 이식은 인슐린 의존형 당뇨병인 제1형 당뇨병에서 정상혈당을 회복시키고, 당뇨 합병증을 방지할 수 있는 치료방법으로 사용되고 있다. 하지만 당뇨병 환자에게 이식을 위한 이자섬의 양에 비해 공여자로부터 증여된 이자섬의 양은 제한적이다. 이러한 문제점은 이자섬의 증식으로 연구되고 있으나, 배양된 이자섬이 정상 조직내의 이자섬과 형태적 기능적으로 동일한 것인지에 관한 연구는 미비하였다. 따라서 본 연구에서는 분리된 이자섬과 배양된 이자섬을 구성하는 세포들의 내부구조의 변화를 주사전자현미경, 투과전자현미경을 이용하여 세포의 미세구조를 확인하고, 인슐린 항체를 이용한 $\beta$세포 내의 인슐린 분포양상을 확인하여 다음과 같은 결과를 얻었다. 분리된 이자섬의 $\beta$세포는 일반적인 핵 미토콘드리아, 세포질세망 그리고 인슐린 과립이 분포하고, 배양된 이자섬 $\beta$세포의 경우 분리된 이자섬에 비하여 일반적인 핵의 모습과 부피가 증가한 세포질과 미토콘드리아, 세포질세망 그리고 골지체의 발달이 이루어지는 것으로 관찰되었다. 인슐린 과립의 경우 분리된 이자섬에 비해 감소하며, 세포막 주위에 분포하는 것으로 관찰되었다. 배양된 이자섬에서 관찰되는 인슐린 과립 분포의 변화, 세포질세망의 증가, 골지체의 발달은 배양된 이자섬 $\beta$세포의 인슐린 생성 분비 기능의 향상과 부피의 증가가 이루어지기 위한 세포 내부의 형태적 변화가 이루어지는 것으로 추측된다.

• 한국결정학회:학술대회논문집
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• 한국결정학회 2003년도 춘계학술연구발표회
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• pp.18-18
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• 2003

Thioredoxin (Trx) is a small redox protein that is ubiquitously distributed from achaes to human. In diverse organisms, the protein is involved in various physiological roles by acting as electron donor and regulators of transcription and apoptosis as well as antioxidants. Sequences of Trx within various species are 27～69% identical to that of E. coli and all Trx proteins have the same overall fold, which consists of central five β strands surrounded by four α helices. The N-terminal cysteine in WCGPC motif of Trx is redox sensitive and the motif is highly conserved. Compared with general cysteine, the N-terminal cysteine has low pKa value. The result leads to increased reduction activity of protein. Recently, novel thio.edoxin-related protein (TRP14) was found from rat brain. TRP14 acts as disulfide reductase like Trx1, and its redox potential and pKa are similar to those of Trx1. However, TRP14 takes up electrons from cytosolic thioredoxin reductase (TrxR1), not from the mitochondrial thioredoxin reductase (TrxR2). Biological roles of TES14 were reported to be involved in regulating TNF-α induced signaling pathways in different manner with Trx1. In depletion experiments, depletion of TRP14 increased TNF-α induced phosphorylation and degradation of IκBα more than the depletion Trx1 did. It also facilitated activation of JNK and p38 MAP kinase induced by TNF-α. Unlike Trx1, TRP14 shows neither interaction nor interference with ASK1. Here, we determined three-dimensional crystal structure of TRP14 by MAD method at 1.8Å. The structure reveals that the conserved cis-Pro (Pro90) and active site-W-C-X-X-C motif, which may be involved in substrate recognition similar to Trx1 , are located at the beginning position of strand β4 and helix α2, respectively. The TRP14 structure also shows that surface of TRP14 in the vicinity of the active site, which is surrounded by an extended flexible loop and an additional short a helix, is different from that of Trx1. In addition, the structure exhibits that TRP14 interact with a distinct target proteins compared with Trx1 and the binding may depend mainly on hydrophobic and charge interactions. Consequently, the structure supports biological data that the TRP14 is involved in regulating TNF-α induced signaling pathways in different manner with Trx1.

• 대한두개안면성형외과학회지
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• 제19권3호
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• pp.181-189
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• 2018

Background: Autogenous bone grafts have several limitations including donor-site problems and insufficient bone volume. To address these limitations, research on bone regeneration is being conducted actively. In this study, we investigate the effects of a three-dimensionally (3D) printed polycaprolactone (PCL)/tricalcium phosphate (TCP) scaffold on the osteogenic differentiation potential of adipose tissue-derived stem cells (ADSCs) and bone marrow-derived stem cells (BMSCs). Methods: We investigated the extent of osteogenic differentiation on the first and tenth day and fourth week after cell culture. Cytotoxicity of the 3D printed $PCL/{\beta}-TCP$ scaffold was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, prior to osteogenic differentiation analysis. ADSCs and BMSCs were divided into three groups: C, only cultured cells; M, cells cultured in the 3D printed $PCL/{\beta}-TCP$ scaffold; D, cells cultured in the 3D printed $PCL/{\beta}-TCP$ scaffold with a bone differentiation medium. Alkaline phosphatase (ALP) activity assay, von Kossa staining, reverse transcription-polymerase chain reaction (RT-PCR), and Western blotting were performed for comparative analysis. Results: ALP assay and von Kossa staining revealed that group M had higher levels of osteogenic differentiation compared to group C. RT-PCR showed that gene expression was higher in group M than in group C, indicating that, compared to group C, osteogenic differentiation was more extensive in group M. Expression levels of proteins involved in ossification were higher in group M, as per the Western blotting results. Conclusion: Osteogenic differentiation was increased in mesenchymal stromal cells (MSCs) cultured in the 3D printed PCL/TCP scaffold compared to the control group. Osteogenic differentiation activity of MSCs cultured in the 3D printed PCL/TCP scaffold was lower than that of cells cultured on the scaffold in bone differentiation medium. Collectively, these results indicate that the 3D printed PCL/TCP scaffold promoted osteogenic differentiation of MSCs and may be widely used for bone tissue engineering.

• Reproductive and Developmental Biology
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• 제30권3호
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• pp.175-180
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• 2006

본 연구는 융합 전 수핵란의 활성화 처리 유무와 활성화 처리 시간이 소 체세포유래 핵이식란의 체외 발육능에 미치는 영향을 검토하기 위해 수행하였다. 소 체외성숙란의 탈핵 후 체세포 핵을 이식하고 일부는 전기 융합 후 활성화를 유기하였고(pre-AC), 일부는 먼저 활성화 처리 후 융합을 실시(post-AC)하였다. 난자의 활성화는 $Ca^{2+}$-ionophore(A23187)로 처리 후 DMAP로 4시간 배양하는 방법으로 유기하였다. 핵이식란은 CR1aa액에서 9일간 배양하여 발육율을 검토하였으며, 활성화 후 30분${\sim}$2.5시간에 고정하여 confocal microscope하에서 핵형 변화를 검사하였다. 배반포기까지 발육율은 post-AC구(20.6%)가 pre-AC(15.3%)보다 다소 높게 나타났다. 또한 활성화 처리를 하여 핵이식란의 배 발달을 비교한 결과 post-AC구가 더 늦은 배 발달 속도를 나타내었다. Post-AC구를 활성화 후 30분, 2시간, 4시간으로 나누어 융합하여 발육율을 검토한 결과 발육율에 차이가 없었다. 본 연구의 결과는 수핵란의 활성화 시간에 따라 핵이식란의 발육 및 핵형이 영향을 받을 수 있음을 시사한다.

• Journal of Veterinary Science
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• 제22권5호
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• pp.63.1-63.14
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• 2021

Background: Recently, mesenchymal stem cells therapy has been performed in dogs, although the outcome is not always favorable. Objectives: To investigate the therapeutic efficacy of mesenchymal stem cells (MSCs) using dog leukocyte antigen (DLA) matching between the donor and recipient in vitro. Methods: Canine adipose-derived MSCs (cA-MSCs) isolated from the subcutaneous tissue of Dog 1 underwent characterization. For major DLA genotyping (DQA1, DQB1, and DRB1), peripheral blood mononuclear cells (PBMCs) from two dogs (Dogs 1 and 2) were analyzed by direct sequencing of polymerase chain reaction (PCR) products. The cA-MSCs were co-cultured at a 1:10 ratio with activated PBMCs (DLA matching or mismatching) for 3 days and analyzed for immunosuppressive (IDO, PTGS2, and PTGES), inflammatory (IL6 and IL10), and apoptotic genes (CASP8, BAX, TP53, and BCL2) by quantitative real-time reverse transcriptase-PCR. Results: cA-MSCs were expressed cell surface markers such as CD90⁺/44⁺/29⁺/45^- and differentiated into osteocytes, chondrocytes, and adipocytes in vitro. According to the Immuno Polymorphism Database, DLA genotyping comparisons of Dogs 1 and 2 revealed complete differences in genes DQA1, DQB1, and DRB1. In the co-culturing of cA-MSCs and PBMCs, DLA mismatch between the two cell types induced a significant increase in the expression of immunosuppressive (IDO/PTGS2) and apoptotic (CASP8/BAX) genes. Conclusions: The administration of cA-MSCs matching the recipient DLA type can alleviate the need to regulate excessive immunosuppressive responses associated with genes, such as IDO and PTGES. Furthermore, easy and reliable DLA genotyping technology is required because of the high degree of genetic polymorphisms of DQA1, DQB1, and DRB1 and the low readability of DLA 88.