• 생명과학회지
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• 제18권10호
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• pp.1348-1354
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• 2008

mi transcription factor (MITF)는 비만세포의 분화를 조절하는 중요한 전사인자이다. 특히 MITF는 결합조직형 비만세포에서 일반적으로 발현하는 비만세포 특이적 세린 단백분해효소의 일종인 mMCP-6 유전자의 전사를 조절한다. 본 연구는 마우스 골수유래 배양비만세포에서 mMCP-6 유전자의 전사를 조절하는 MITF이형체를 규명하였다. MITF 이형체들의 발현은 RT-PCR로 확인하였다. IL-3존재 하에서 배양한 점막형 비만세포들은 MITF-A,-E, -H, -Mc 등이 발현하였다. 반면에 SCF존재 하에서 배양한 결합조직형 비만세포들은 MITF-A가 발현하였다. MITF이형체를 과발현시키면 NIH-3T3 세포에서 mMCP-6 promoter를 통한 luciferase 활성을 증가시키고, MC/9 비만세포주에서는 증가된 mMCP-6발현을 유도하였다. 더불어 비만세포에서의 mMCP-6 발현은 MITF-A 고갈로 인하여 유의적으로 억제되었다. MITF-A의 전사활성과 DNA결합은 MITF-E, -H, -Mc 등의 타 이형체들의 결과와 유사하였다. 따라서 본 연구의 결과들은 MITF-A가 마우스 결합조직형 비만세포에서 발현하여 mMCP-6 전사를 조절하는 중요한 이형체임을 제시한다.

• Reproductive and Developmental Biology
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• 제36권1호
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• pp.7-12
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• 2012

DNA methyltransferase 1 (Dnmt1) gene contains three different isoform transcripts, Dnmt1s, Dnmt1o, and Dnmt1p, are produced by alternative usage of multiple first exons. Dnmt1o is specific to oocytes and preimplantation embryos, whereas Dnmt1s is expressed in somatic cells. Here we determined that porcine Dnmt1o gene had differentially methylated regions (DMRs) in 5'-flanking region, while those were not found in the Dnmt1s promoter region. The methylation patterns of the porcine Dnmt1o/Dnmt1s DMRs were investigated using bisulfite sequencing and pyrosequencing analysis through all preimplantation stages from one cell to blastocyst stage in in vivo or somatic cell nuclear transfer (SCNT). The Dnmt1o DMRs contained 8 CpG sites, which located in -640 bp to -30 bp upstream region from transcription start site of the Dnmt1o gene. The methylation status of 5 CpGs within the Dnmt1o DMRs were distinctively different at each stage from one-cell to blastocyst stage in the $in$ $vivo$ or SCNT, respectively. 55.62% methylation degree of the Dnmt1o DMRs in the $in$ $vivo$ was increased up to 84.38% in the SCNT embryo, moreover, $de$ $novo$ methylation and demethylation occurred during development of porcine embryos from the one-cell stage to the blastocyst stage. However, the DNA methylation states at CpG sites in the Dnmt1s promoter regions were hypomethylated, and dramatically not changed through one-cell to blastocyst stage in the $in$ $vivo$ or SCNT embryos. In the present study, we demonstrated that the DMRs in the promoter region of the porcine Dnmt1o was well conserved, contributing to establishment and maintenance of genome-wide patterns of DNA methylation in early embryonic development.

• Journal of Microbiology and Biotechnology
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• 제33권3호
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• pp.329-338
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• 2023

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that produces attaching and effacing lesions on the large intestine and causes hemorrhagic colitis. It is primarily transmitted through the consumption of contaminated meat or fresh produce. Similar to other bacterial pathogens, antibiotic resistance is of concern for EHEC. Furthermore, since the production of Shiga toxin by this pathogen is enhanced after antibiotic treatment, alternative agents that control EHEC are necessary. This study aimed to discover alternative treatments that target virulence factors and reduce EHEC toxicity. The locus of enterocyte effacement (LEE) is essential for EHEC attachment to host cells and virulence, and most of the LEE genes are positively regulated by the transcriptional regulator, Ler. GrlA protein, a transcriptional activator of ler, is thus a potential target for virulence inhibitors of EHEC. To identify the GrlA inhibitors, an in vivo high-throughput screening (HTS) system consisting of a GrlA-expressing plasmid and a reporter plasmid was constructed. Since the reporter luminescence gene was fused to the ler promoter, the bioluminescence would decrease if inhibitors affected the GrlA. By screening 8,201 compounds from the Korea Chemical Bank, we identified a novel GrlA inhibitor named Grlactin [3-[(2,4-dichlorophenoxy)methyl]-4-(3-methylbut-2-en-1-yl)-4,5-dihydro-1,2,4-oxadiazol-5-one], which suppresses the expression of LEE genes. Grlactin significantly diminished the adhesion of EHEC strain EDL933 to human epithelial cells without inhibiting bacterial growth. These findings suggest that the developed screening system was effective at identifying GrlA inhibitors, and Grlactin has potential for use as a novel anti-adhesion agent for EHEC while reducing the incidence of resistance.

• Asian-Australasian Journal of Animal Sciences
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• 제19권3호
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• pp.425-430
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• 2006

Three hundred and sixty sexed 3-day-old broiler chicks were divided randomly into six treatment groups (control, antibiotic and black cumin at four levels) of 60 birds each. Black cumin seeds at 0.5%, 1%, 2% or 3% and avilamycin at 10 mg/kgt were added to the basal diet and their effects determined on feed intake, daily live weight gain, feed conversion ratio and carcass characteristics. There were no significant differences in daily feed intake at 21 and 42 days (p>0.05). Average daily gain was significantly different between the treatments. The birds fed the diet containing 1% black cumin seeds and antibiotic were the highest average daily gain, followed by those the other treatment diets and negative control (p<0.05). From 1 to 42 days of age, feed conversion ratios were improved significantly by supplementation with 1% black cumin seeds and with antibiotic (p<0.05) by approximately 5% compared to the control group. Similarly, the highest cold carcass, thigh, breast, wing, neck and liver weights were observed in the 1% black cumin and antibiotic groups (p<0.05). Accordingly, 1% supplementation of black cumin seeds to diets could be considered as an alternative natural growth promoter for poultry instead of antibiotics.

• Journal of Plant Biotechnology
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• 제36권4호
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• pp.309-319
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• 2009

Transgenic plant cell cultures for the production of biopharmaceuticals including monoclonal antibodies, recombinant proteins have been regarded as an alternative platform in addition to traditional microbial fermentation and mammalian cell cultures. Plant-made pharmaceuticals (PMPs) have several advantages such as safety, cost-effectiveness, scalability and possibility of complex post-translational modifications. Increasing demand for the quantity and diversity of pharmaceutical proteins may accelerate the industrialization of PMP technology. Up to date, there is no plant-made recombinant protein approved by USFDA (Food and Drug Administration) for human therapeutic uses due to the technological bottlenecks of low expression level and slight differences in glycosylation. Regarding expression levels, it is possible to improve the productivity by using stronger promoter and optimizing culture processes. In terms of glycosylation, humanization has been attempted in many ways to reduce immune responses and to enhance the efficacy as well as stability. In this review article, all these respects of transgenic plant cell cultures were summarized. In addition, we also discuss the general characteristics of plant cell suspension cultures related with bioreactor design and operation to achieve high productivity in large scale which could be a key to successful commercialization of PMPs.

• Molecules and Cells
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• 제25권3호
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• pp.446-451
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• 2008

We assessed heterologous protein expression in 64 strains obtained from the Escherichia coli Reference (ECOR) collection, a collection representing diverse natural E. coli populations. A plasmid generating a glutathione S-transferase and plant carbonic anhydrase fusion protein (GST-CA) under the control of the tac promoter was introduced into the ECOR strains, and the quantity of the fusion protein was determined by SDS-PAGE. The foreign protein was generated at various levels, from very high (40 strains, high producers) to very low (six strains, low producers). Immunoblotting showed that the high producers expressed approximately 250-500 times more GST-CA protein than the low producers. The results of semi-quantitative RT-PCR showed that the low producers generated mRNA levels comparable to those of the high producers, thereby suggesting that, at least in this case, inefficient translation is a major cause of the low production. We introduced a different plasmid, which expressed a maltose binding protein and plant guanylate kinase fusion protein (MBP-GK) into the six low producers. Interestingly, five of these expressed MBP-GK at very high levels. Thus, we conclude that the production of a particular protein from an expression vector can vary considerably, depending on the host strain. Strains in the ECOR collection could function as useful alternative hosts when a desired level of protein expression is not obtained from commonly used strains, such as E. coli K12 or B derivatives.

• 한국축산식품학회지
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• 제36권5호
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• pp.567-576
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• 2016

Poultry industry has always been a dynamic and integral part of national economies in many countries. Economic losses incur especially in large-scale rearing facilities, often attributed to the deterioration of environmental conditions, poultry exposure to stressors and development of diseases. While antibiotics have been commonly used for prophylactic purposes and as growth stimulants, extensive documentation of antimicrobial resistance among pathogenic bacteria due to indiscriminate utilization of antibiotic in the industry has led to public and governmental outcries. Elimination of antibiotics from poultry production has thus encouraged intensive search for alternatives. In this review, we discuss the immense potential of probiotics to fill the gap as alternative growth promoters and evidences of beneficial effects of probiotic application in poultry production.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.52-52
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• 2003

Junctate is a newly identified integral ER/SR membrane $Ca^{2+}$ binding protein, which is an alternative splicing form of the same gene generating aspartyl $\square$-hydroxylase and junctin. To elucidate the functional role of junctate in heart, transgenic (TG) mice overexpressing mouse cardiac junctate-1 under the control of mouse $\square$$^{~}$ myosin heavy chain promoter were generated. Overexpression of junctate in mouse heart resulted in cardiac hypertrophy, increased fibrosis, bradycardia, arrhythmias and impaired contractility. Overexpression of junctate also led to down-regulation of SERCA2, calsequestrin, calreticulin and RyR, but to up-regulation of NCX and PMCA. The SR $Ca^{2+}$ content decreased and the L-type $Ca^{2+}$ current density and the action potential durations increased in TG cardiomyocytes, which could be the cause for the bradycardia in TG heart. The present work has provided an important example of pathogenesis leading to cardiac hypertrophy and arrhythmia, which was caused by impaired $Ca^{2+}$ handling by overexpression of junctate in heart.n heart.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제36권6호
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• pp.481-489
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• 2010

Introduction: TLR-5, a member of the toll-like receptor (TLR) family, is a element of the type I transmembrane receptors, which are characterized by an intracellular signaling domain homolog to the interleukin-1 receptor. These receptors recognize microbial components, particularly bacterial flagellin. All-trans retinoic acid (atRA, tretinoin), a natural metabolite of vitamin A, acts as a growth and differentiation factor in many tissues, and is also needed for immune functions. In this study, THP-1 human macrophage-monocytes were used to examine the mechanisms by which atRA regulated the expression of TLR-5. Because the molecular mechanism underlying this regulation at the transcriptional level is also unclear, this study examined which putative transcription factors are responsible for TLR-5 expression by atRA in immune cells. Materials and Methods: This study examined whether atRA induces the expression of TLR-5 in THP-1 cells using reverse transcription-polymerase chain reaction (RT-PCR), and which transcription factors are involved in regulating the TLR-5 promoter in RAW264.7 cells using a reporter assay system. Western blot analysis was used to determine which signal pathway is involved in the expression of TLR-5 in atRA-treated THP-1 cells. Results: atRA at a concentration of 10 nM greatly induced the expression of TLR-5 in THP-1 cells. Human TLR-5 promoter contains three Sp-1/GC binding sites around -50 bp and two NF-kB binding sites at -380 bp and -160 bp from the transcriptional start site of the TLR-5 gene. Sp-1/GC is primarily responsible for the constitutive TLR-5 expression, and may also contribute to NF-kB at -160 bp to induce TLR-5 after atRA stimulation in THP-1 cells. The role of NF-kB in TLR-5 expression was further confirmed by inhibitor pyrrolidine dithiocarbamate (PDTC) experiments, which greatly reduced the TLR-5 transcription by 70-80%. Conclusion: atRA induces the expression of the human TLR-5 gene and NF-kB is a critical transcription factor for the atRA-induced expression of TLR-5. Accordingly, it is conceivable that retinoids are required for adequate innate and adaptive immune responses to agents of infectious diseases. atRA and various synthetic retinoids have been used therapeutically in human diseases, such as leukemia and other cancers due to the antiproliferative and apoptosis inducing effects of retinoids. Therefore, understanding the molecular regulatory mechanism of TLR-5 may assist in the design of alternative strategies for the treatment of infectious diseases, leukemia and cancers.

• 한국수정란이식학회지
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• 제33권3호
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• pp.129-137
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• 2018

Acute vascular rejection has been known as a main barrier occurring in a xenograted tissue of alpha 1,3-galactosyltransferase knock-out (GalT KO) pig into a non-human primate (NHP). Adenosine which is a final metabolite following sequential hydrolysis of nucleotide by ecto-nucleotidases such as CD39 and CD73, act as a regulator of coagulation, and inflammation. Thus xenotransplantation of CD39 and CD73 expressing pig under the GalT KO background could lead to enhanced survival of recipient NHP. We constructed a human CD39 and CD73 expression cassette designed for endothelial cell-specific expression using porcine Icam2 promoter (pIcam2-hCD39/hCD73). We performed isolation of endothelial cells (pAEC) from aorta of 4 week-old GalT KO and membrane cofactor protein expressing pig ($GalT^{-MCP/-MCP}$). We were able to verify that isolated cells were endothelial-like cells using immunofluorescence staining analysis with von Willebrand factor antibody, which is well known as an endothelial maker, and tubal formation assay. To find optimal condition for efficient transfection into pAEC, we performed transfection with GFP expression vector using four programs of nucleofection, M-003, U-023, W-023 and Y-022. We were able find that the program W-023 was optimal for pAEC with regard to viability and transfection efficiency by flow cytometry and fluorescent microscopy analyses. Finally, we were able to obtain $GalT^{-MCP/-MCP}/CD39/CD73$ pAEC expressing CD39 and CD73 at levels of 33.3% and 26.8%, respectively. We suggested that pACE isolated from $GalT^{-MCP/-MCP}$ pig might be provided as a basic resource to understand biochemical and molecular mechanisms of the rejections and as an alternative donor cells to generate $GalT^{-MCP/-MCP}/CD39/CD73$ pig expressing CD39 and CD73 at endothelial cells.