• Asian-Australasian Journal of Animal Sciences
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• 제33권2호
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• pp.219-229
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• 2020

Objective: Considering the physiological and clinical importance of leptin receptor (LEPR) in regulating obesity and the fact that porcine LEPR expression is not known to be controlled by lncRNAs and miRNAs, we aim to characterize this gene as a potential target of SSC-miR-323 and the lncRNA TCONS_00010987. Methods: Bioinformatics analyses revealed that lncRNA TCONS_00010987 and LEPR have SSC-miR-323-binding sites and that LEPR might be a target of lncRNA TCONS_00010987 based on cis prediction. Wild-type and mutant TCONS_00010987-target sequence fragments and wild-type and mutant LEPR 3'-UTR fragments were generated and cloned into pmiRRB-REPORT^TM-Control vectors to construct respective recombinant plasmids. HEK293T cells were co-transfected with the SSC-miR-323 mimics or a negative control with constructs harboring the corresponding binding sites and relative luciferase activities were determined. Tissue expression patterns of lncRNA TCONS_00010987, SSC-miR-323, and LEPR in Anqing six-end-white (AQ, the obese breed) and Large White (LW, the lean breed) pigs were detected by real-time quantitative polymerase chain reaction; backfat expression of LEPR protein was detected by western blotting. Results: Target gene fragments were successfully cloned, and the four recombinant vectors were constructed. Compared to the negative control, SSC-miR-323 mimics significantly inhibited luciferase activity from the wild-type TCONS_00010987-target sequence and wild-type LEPR-3'-UTR (p<0.01 for both) but not from the mutant TCONS_00010987-target sequence and mutant LEPR-3'-UTR (p>0.05 for both). Backfat expression levels of TCONS_00010987 and LEPR in AQ pigs were significantly higher than those in LW pigs (p<0.01), whereas levels of SSC-miR-323 in AQ pigs were significantly lower than those in LW pigs (p<0.05). LEPR protein levels in the backfat tissues of AQ pigs were markedly higher than those in LW pigs (p<0.01). Conclusion: LEPR is a potential target of SSC-miR-323, and TCONS_00010987 might act as a sponge for SSC-miR-323 to regulate LEPR expression.

• 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.

• 생명과학회지
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• 제18권4호
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• pp.530-536
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• 2008

본 연구는 재조합 단백질 생산에 흔히 사용되는 P. fluorescens에 대한 공기 중 노출 시 염증작용이 의심되는 기전을 분석하기 위하여 인체 폐포 상피세포에서의 염증성 인자들 특히 IL-8, COX-2, MIC-1의 발현을 분석하고자 하였다. 균주 P. fluorescens에 대한 인체 폐포 상피세포 A549에서의 세포증식 억제효과를 밝혔고, 상피세포의 염증성 사이토카인으로서 대표적인 인터루킨 8의 발현에 대해서 분석결과. 균주 P. fluorescens및 재조합 균주에 의해 IL-8의 분비가 진핵세포대비 세균세포숫자 의존적으로 생성량이 증대되었다. 또한 점막 상피세포의 염증성 프로스타글란딘 생성에서 핵심적인 역할을 하는 cyclooxygenase-2 (COX-2)에 대해서 P. fluorescens 는 COX-2의 mRNA 발현이 소량 증진되었으나 실제 단백질의 양 및 전사의 변화는 없었다. 일반적으로 단핵구의 염증성 사이토카인 생성에 대하여 억제성 작용을 하는 macrophage inhibitory cytokine 1 (MIC-1)의 발현에 대해서 측정결과 유전자의 발현이 증대되는 효과는 미비하였으나, MIC-1 단백질의 processing에서 propeptide (${\sim}28\;kD$) 및 mature MIC-1 (${\sim}15\;kD$)로 분해 되어 MIC-1의 단백질 활성화가 증대되었다. 본 연구에서 보이는 MIC-1은 P. fluorescens에 의한 세포 생존율 억제작용에도 기여할 것으로 예측되며, IL-8에 의한 염증구의 recruitment 대한 억제작용이 예상되나, MIC-1의 활성이 과도하고 지속적으로 나타날 경우 조직의 손상도 가능성도 있다.

• IMMUNE NETWORK
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• 제8권1호
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• pp.21-28
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• 2008

Background: A co-inhibitory molecule, B7-H4, is believed to negatively regulate T cell immunity by suppressing T cell proliferation and inhibiting cytokine production. However, the mechanism behind B7-H4-mediated tolerance remains unclear. Methods: Balb/c $(H-2^d)$ mice were fed with dendritic cell line, DC2.4 $(H-2^d)$ every day for 10 days. Meantime, mice were hydrodynamically injected with recombinant plasmid expressing B7-H4 fusion protein (B7-H4.hFc) or hFc via tail vein. One day after last feeding, mice were immunized with allogeneic B6 spleen cells. 14 days following immunization, mice were challenged with B6 spleen cells to ear back and the ear swelling was determined the next day. Subsequently, a mixed lymphocyte reaction (MLR) was also performed and cytokines profiles from the reaction were examined by sandwich ELISA. Frequency of immunosuppressive cell population was assayed with flow cytometry and mRNA for FoxP3 was determined by RT-PCR. Results: Tolerant mice given plasmid expressing B7-H4.hFc showed a significant reduction in ear swelling compared to control mice. In addition, T cells from mice given B7-H4.hFc plasmid revealed a significant hyporesponsiveness of T cells against allogeneic spleen cells and showed a significant decrease in Th1 and Th2 cytokines such as IFN-${\gamma}$, IL-5, and TNF-${\alpha}$. Interestingly, flow cytometric analysis showed that the frequency of CD4+CD25+FoxP3+ Tregs in spleen was increased in tolerant mice given recombinant B7-H4.hFc plasmid compared to control group. Conclusion: Our results demonstrate that B7-H4 synergistically potentiates oral tolerance induced by allogeneic cells by increasing the frequency of FoxP3+ CD4+CD25+ Treg and reducing Th1 and Th2 cytokine production.

• 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.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2001년도 추계학술대회
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• pp.46-53
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• 2001

A commensal thermophile, Symbiobacterium toebii, isolated from hay compost (toebii) in Korea commensally interacted with a thermophilic Geobacillus toebii sp. nov., which was a new species within the genus Geobacillus on the basis of the phenotypic traits and molecular systematic data. S. toebii required the crude extracts and/or culture supernatant of the Geobacillus toebii for axenic growth and could grow on the temperature between 45 and $70^{\circ}C$ (optimum: $60^{\circ}C$; 2.4 h doubling time) and pH 6.0 and 9.0 (optimum: pH 7.5). The G+C content of the genomic DNA was $65 mol\%$, and the major quinones were MK-6 and MK-7. A phylogenetic analysis of its 16S rDNA sequence indicated that Symbiobacterium toebii was closely related with solely reported Symbiobacterium thermophilum. The presence of the commensal thermophile 16S rDNA and accumulation of indole in all the enriched cultures indicate that Symbiobacterium toebii is widely distributed in the various soils. The genome of S. toebii constituted a circular chromosome of 3,280,275 base pairs and there was not an extra-chromosomal element (ECE). It contained about 4,107 predicted coding sequences. Of these protein coding genes, about $45.6\%$ was encoded well-known proteins and annotated the functional assignment of 1,874 open reading frames (ORFs), and the rest predicted to have unknown functions. The genes encoding thermostable tyrosine phenol-lyase and tryptophan indole-lyase were cloned from the genomic DNA of S. toebii and the enzymatic production of L-tyrosine and L-tryptophan was carried out with two thermostable enzymes overexpressed in recombinant E. coli.

• 대한의생명과학회지
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• 제9권4호
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• pp.177-181
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• 2003

The baculovirus/Spodoptera frugiperda (Sf) cell system has become popular for the production of large amounts of the human erythrocyte glucose transporter, GLUT1, heterologously. However, it was not possible to show that the expressed transporter in insect cells could actually transport glucose. The possible reason for this was that the activity of the endogenous insect glucose transporter was extremely high and so rendered transport activity resulting from the expression of exogenous transporter very difficult to detect. Sf21-AE cells are commonly employed as the host permissive cell line to support the baculovirus AcNPV replication and protein synthesis. The cells grow well on TC-100 medium that contains 0.1 % D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, unlike the human glucose transporter, very little is known about properties of the endogenous sugar transporter(s) in insect cells. Thus, the uptake of 2-deoxy-D-glucose (2dGlc) by Sf21-AE cells and the inhibition of 2dGlc transport in the insect cells by fructose and cytochalasin B were investigated in the present work. The binding assay of cytochalasin B was also performed, which could be used as a functional assay for the endogenous glucose transporter(s) in the insect cells. Sf21-AE cells were infected with the recombinant virus AcNPV-GT or no virus, at a multiplicity of infection (MOI) of 5. Infected cells were resuspended in PBS plus and minus 300 mM fructose, and plus and minus 20 $\mu$M cytochalasin B for use in transport assays. Uptake was measured at 28$^{\circ}C$ for 1 min, with final concentration of 1 mM deoxy-D-glucose, 2-[1,2-$^3$H]- or glucose, L-[l,$^3$H]-, used at a specific radioactivity of 4 Ci/mol. The results obtained demonstrated that the sugar uptake in uninfected cells was stereospecific, and was strongly inhibited by fructose but only poorly inhibitable by cytochalasin B. It is therefore suggested that the Sf21-AE glucose transporter has very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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• pp.1239-1248
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• 2004

The methylotrophic yeast Hansenula polymorpha has been extensively studied as a model organism for methanol metabolism and peroxisome biogenesis. Recently, this yeast has also attracted attention as a promising host organism for recombinant protein production. Here, we describe the fabrication and evaluation of a DNA chip spotted with 382 open reading frames (ORFs) of H. polymorpha. Each ORF was PCR-amplified using gene-specific primer sets, of which the forward primers had 5'-aminolink. The PCR products were printed in duplicate onto the aldehyde-coated slide glasses to link only the coding strands to the surface of the slide via covalent coupling between amine and aldehyde groups. With the partial genome DNA chip, we compared efficiency of direct and indirect cDNA target labeling methods, and found that the indirect method, using fluorescent-labeled dendrimers, generated a higher hybridization signal-to-noise ratio than the direct method, using cDNA targets labeled by incorporation of fluorescence-labeled nucIeotides during reverse transcription. In addition, to assess the quality of this DNA chip, we analyzed the expression profiles of H. polymorpha cells grown on different carbon sources, such as glucose and methanol, and also those of cells treated with the superoxide­generating drug, menadione. The profiles obtained showed a high-level induction of a set of ORFs involved in methanol metabolism and oxidative stress response in the presence of methanol and menadione, respectively. The results demonstrate the sensitivity and reliability of our arrays to analyze global gene expression changes of H. polymorpha under defined environmental conditions.

• 한국미생물·생명공학회지
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• 제37권4호
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• pp.355-360
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• 2009

P. carageenovora 유래 arylsulfatase 유전자(astA)의 효모표면발현 plasmid pCTAST(7.1 kb)를 구축하여 S. cerevisiae EBY100에 형질전환하였고, 선별된 형질전환체들을 YPDG 배지에서 배양했을 때 4-methylumbelliferyl sulfate를 분해하여 형광을 보였다. 이는 효모에서 arylsulfatase가 활성형으로 생산되었음을 의미하였다. S. cerevisiae EBY100/pCTAST를 플라스크 배양했을 때 arylsulfatase 활성은 galactose가 완전히 소모된 배양 48시간째 최대 활성인 1.2 unit/mL로 나타났으며 배양 상등액에서는 활성이 나타나지 않았다. 효모 S. cerevisiae의 세포표면에서 발현된 재조합 arylsulfatase로 제조된 agarose를 agar와 시판용 agarose와 함께 ${\lambda}DNA$ HindIII marker를 사용하여 DNA 전기영동 성능을 비교 실험한 결과, agar보다는 재조합 arylsulfatase 처리로 제조한 agarose가 이동성이나 분리능에서 우수하였으며, 시판용 agarose와 비교하여 이동성이나 분리능이 유사한 결과를 확인할 수 있었다. 본 연구의 결과, 효모 S. cerevisiae의 세포표면에서 발현된 재조합 arylsulfatase 효소를 이용하여 한천으로부터 전기영동용 고순도 친환경적인 agarose 생물 생산 공정에 적용 가능함을 알 수 있었다.

• 대한소아치과학회지
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• 제35권1호
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• pp.73-82
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• 2008

치아우식은 주로 mutans streptococci에 의해 야기되는 감염성 질환으로서 주 원인균에는 streptococcus mutans가 있다. S. mutans가 치아우식을 유발하는 분자 생물학적 기전은 몇 가지 단계를 포함한다. 먼저 S. mutans는 AgI/II와 같은 세포 표면의 섬유성 단백질을 매개로 치면의 타액성 피막에 일차적으로 부착한다. 두번째 단계에서 자당의 존재하에 glucosyltransferase(GTF)는 glucan과 같은 다당체를 합성하게 된다. 마지막으로 이렇게 합성된 glucan은 glucan binding proteins와 상호작용하여 치면세균막을 형성해서 세균의 군집화를 가능하게 한다. 많은 실험과 임상연구에서 S. mutans의 주요 항원(Ag I/II, GTFs, GBPs)들이 치아우식 병리기전에 영향을 준다고 알려져 왔고, 따라서 이런 항원들이 면역계에 작용하여 치아우식을 막는 백신으로 이용 가능하다. 본 실험은 streptococcus mutans GS-5로부터, GTFb, GTFc, GTFd 유전자를 복제하고 염기서열분석을 하였으며, 이중 GTFd가 먼저 재조합 단백질 생산을 위해 발현 벡터에 클로닝 되었으며, 이로부터 단백질이 발현됨을 확인하였다. 이번 실험에서 얻은 순수 GTF 항원은 동물실험을 통해 특정 GTF 활성부위에 대한 항체 생산에 이용될 수 있을 것이다.