• BMB Reports
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• 제43권3호
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• pp.193-198
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• 2010

In this study, we report the identification and characterization of a novel C2H2 zinc finger protein, ZNF552, from a human embryonic heart cDNA library. ZNF552 is composed of three exons and two introns and maps to chromosome 19q13.43. The cDNA of ZNF552 is 2.3 kb, encoding 407 amino acids with an amino-terminal KRAB domain and seven carboxyl-terminal C2H2 zinc finger motifs in the nucleus and cytoplasm. Northern blotting analysis indicated that a 2.3 kb transcript specific for ZNF552 was expressed in liver, lung, spleen, testis and kidney, especially with a higher level in the lung and testis in human adult tissues. Reporter gene assays showed that ZNF552 was a transcriptional repressor, and overexpression of ZNF552 in the COS-7 cells inhibited the transcriptional activities of AP-1 and SRE, which could be relieved through RNAi analysis. Deletion studies showed that the KRAB domain of ZNF552 may be involved in this inhibition.

• Archives of Pharmacal Research
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• 제25권3호
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• pp.364-369
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• 2002

Hepatitis C virus (HCV) is remarkably efficient at establishing chronic infection. One of the reasons for this appears to be the suppression of the accessory cell function of professional antigen presenting cells. In the present study, the immunosuppressive activity of HCV protein was examined on dendritic cells (DCs) generated from mouse bone marrow progenitor cells in vitro. We found that the DCs forced to express HCV protein have defective allostimulatory ability. DCs expressing HCV protein were phenotypically indistinguishable from normal DCs. However, they were unable to produce IL-12 effectively when stimulated with lipopolysaccharide. The functional domain of the HCV protein essential for immunosuppression was determined using a series of ${NH_2}-and$ C-terminal deletion mutants of HCV core protein. We found that amino acid residues residing between the 21 st and the 40th residues from the ${NH_2}-terminus$ of HCV core protein are required for immunosuppression. These findings suggest that HCV core protein suppresses the elicitation of protective Th1 responses by the inhibition of IL-12 production by DCs.

• 한국가축번식학회지
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• 제20권4호
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• pp.453-458
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• 1997

형질전환 동물의 후손에서 transgene은 멘델의 법칙에 따라 유전된다고 일반적으로 인식되어져 왔다. 따라서 본 연구에서는 transgene이 이러한 인식과 일치하는지를 여러 세대를 통하여 확인하고 후손에서 어떻게 유전되는지를 연구하기 위하여 형질전환 생쥐를 생산하여 본 연구의 모델로 삼았다. 수정된 생쥐의 embryo에 DNA를 microinjection하는 방법으로 MMTV-LTR (long terminal repeat), bovine ($\alpha$s1-casein cDNA, 그리고 SV 40 splicing과 polyadenylation site 등의 sequence를 포함한 3.0Kb의 DNA가 주입되었다. 여기에서 태어난 새끼는 dot blot과 Southern blot에 의하여 transgene의 존재여부가 확인되어 founder line이 만들어졌다. 그들의 자손은 PCR에 의해서 transgene이 유전되는지를 확인하였다. F0의 72마리 새끼중에서 4마리의 Founder가 transgene을 가지고 있었다(5.6%). F0에서 F1으로의 유전(transmission)은 각각 33.3, 7.7, 0, 62.5%이었다. Transgene은 F1에서 F2로 각각 63.6, 5.9, 68.8% 유전되었고, F2에서 F3로 각각 85.7, 0, 88.2% 유전되었다. 따라서 본 연구 모델에 의하면 transgene은 멘델의 법칙을 따르는 경우와 deletion이 되는 경우로 각각 관찰되었다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.54-54
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• 2001

Human chorionic gonadotropin (hCG) is a member of the glycoprotein hormone family which includes FSH, hCG, TSH. These hormone family is characterized by a heterodimeric structure composed a common $\alpha$-subunit noncovalently linked to a hormone specific $\beta$-subunit. The correct conformation of the heterodimer is also important for efficient secretion, hormone-specific post-translational modifications, receptor binding and signal transduciton. To determine $\alpha$ and $\beta$-subunits can be synthesized as a single polypeptide chain (tethered-hCG) and also display biological activity, the tethered-hCG molecule by fusing the carboxyl terminus of the hCG $\beta$-subunit to the amino terminus of the $\alpha$-subunit was constructed and transfected into chinese hamster ovary (CHO-K1) cells. We also constructed C-terminal deletion mutants (D9l, D89, D88, D87, D86, D84, D83) of single chain hCG to determine the biological function (secretion, LH-activity, receptor binding, cAMP production) of these mutants. Between six and eight stably transfected pools of cells expressing wild type and mutant hCGs were selected for neomycin resistant. The hCGs secreted by the stably transfected cells into serum-free media were collected and quantified by radioimmunoassay, as described in protocol (DPC(hCG IRMA). LH activity was in terms of testosterone production and aromatase activity in primary cultured rat Leydig cells. The tethered-wthCG was efficiently secreted and showed similar LH-like activity to the dimeric hCG. The D83hCG mutant was not detected in this assay. It is suggest that hCG C-terminal part is very important for hCG secretion. Now, we checking the LH-like activity of these mutant hCGs. These data indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion.

• Molecules and Cells
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• 제28권6호
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• pp.553-558
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• 2009

In the periphery, a galectin-1 receptor, CD7, plays crucial roles in galectin-1-mediated apoptosis of activated T-cells as well as progression of T-lymphoma. Previously, we demonstrated that $NF-{\kappa}B$ downregulated CD7 gene expression through the p38 MAPK pathway in developing immature thymocytes. However, its regulatory pathway is not well understood in functional mature T-cells. Here, we show that CD7 expression was downregulated by Twist2 in Jurkat cells, a human acute T-cell lymphoma cell line, and in EL4 cells, a mature murine T-cell lymphoma cell line. Furthermore, ectopic expression of Twist2 in Jurkat cells reduced galectin-1-induced apoptosis. While full-length Twist2 decreased CD7 promoter activity, a C-terminal deletion form of Twist2 reversed its inhibition, suggesting an important role of the C-terminus in CD7 regulation. In addition, CD7 expression was enhanced by histone deacetylase inhibitors such as trichostatin A and sodium butyrate, which indicates that Twist2 might be one of candidate factors involved in histone deacetylation. Based on these results, we conclude that upregulation of Twist2 increases the resistance to galectin-1-mediated-apoptosis, which may have significant implications for the progression of some T-cells into tumors such as Sezary cells.

• The Korean Journal of Physiology and Pharmacology
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• 제23권3호
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• pp.219-227
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• 2019

Polycystic kidney disease 2-like-1 (PKD2L1), polycystin-L or transient receptor potential polycystin 3 (TRPP3) is a TRP superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. Although the calmodulin (CaM) inhibitor, calmidazolium, is an activator of the PKD2L1 channel, the activating mechanism remains unclear. The purpose of this study is to clarify whether CaM takes part in the regulation of the PKD2L1 channel, and if so, how. With patch clamp techniques, we observed the current amplitudes of PKD2L1 significantly reduced when co-expressed with CaM and $CaM{\triangle}N$. This result suggests that the N-lobe of CaM carries a more crucial role in regulating PKD2L1 and guides us into our next question on the different functions of two lobes of CaM. We also identified the predicted CaM binding site, and generated deletion and truncation mutants. The mutants showed significant reduction in currents losing PKD2L1 current-voltage curve, suggesting that the C-terminal region from 590 to 600 is crucial for maintaining the functionality of the PKD2L1 channel. With PKD2L1608Stop mutant showing increased current amplitudes, we further examined the functional importance of EF-hand domain. Along with co-expression of CaM, ${\triangle}EF$-hand mutant also showed significant changes in current amplitudes and potentiation time. Our findings suggest that there is a constitutive inhibition of EF-hand and binding of CaM C-lobe on the channel in low calcium concentration. At higher calcium concentration, calcium ions occupy the N-lobe as well as the EF-hand domain, allowing the two to compete to bind to the channel.

• 동의생리병리학회지
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• 제20권3호
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• pp.651-656
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• 2006

Activation of $NF-{\kappa}B$ is known to be a trigger of various cellular disorders including inflammatory and autoimmune diseases such as rheumatoid arthritis. Numerous approaches are ongoing within laboratories to identify potential therapeutic agents which inhibit the $NF-{\kappa}B$ activation. In this study, we have tested the inhibitory effects of five traditional medicines on the activation of $NF-{\kappa}B$ by NIK. Among three medicines which exhibited inhibitory effect on the expression of $NF-{\kappa}B$ repoter plasmid, we investigated further the inhibitory mechanism of Dichroa febrifuga in connection with IKKY activity. Wild type $IKK{\gamma}$ inhibited the $NF-{\kappa}B$ activation by NIK but the C-terminal deletion mutant of IKKY did not show the inhibitory effect, indicating that the C-terminal leucine zipper domain of $NF-{\kappa}B$ is important for the inhibition of $NF-{\kappa}B$ activation. The water extract of Dichroa febrifuga(DFE) also strongly inhibited the $NF-{\kappa}B$ activation by NIK. The inhibitory activity of DFE appeared to be independent of the expression of $IKK{\gamma}$, suggesting that the pathways of inhibition by Dichroa febrifuga and $IKK{\gamma}$ are different. Our results suggest that Dichroa febrifuga can be used as a medicine for inhibition of the $NF-{\kappa}B$ activation in a wide range of cells without relation to the expression of $IKK{\gamma}$.

• KSBB Journal
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• 제20권1호
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• pp.21-25
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• 2005

흔하게 사용되어온 제초제인 paraquat는 파킨슨병의 원인이 될 수 있는 유력한 위험 요소이다. 헴산화효소-1(HO-1)은 산화적 스트레스와 소포체 스트레스의 marker인데, 여러 가지 자극에 의해 heme을 분해하여 biliverdin, 일산화탄소, 철 성분으로 전환시킨다 본 연구에서는 뇌의 흑색질 유래의 도파민 세포주 SN4741에서 paraquat가 시간별, 농도별로 HO-1을 활성화시키는 기작을 조사하였다. HO-1이 Paraquat에 의해 활성화되는 것은 주로 유전자 전사 수준에서 조절되었다. HO-1 유전자의 promoter와 5' enhancer인 El, E2를 결실시킨 실험에서, E2 enhancer가 도파민 세포에서 paraquat에 의한 HO-1 유전자 발현을 유도하는 핵심 부위로 판명되었다 E2 enhancer 부위를 돌연변이 시킨 실험 결과는 전사인자 활성 단백질-1 (AP-1) 결합부위를 통해 HO-1 발현이 유도됨을 밝히게 되었다. 또한, 도파민 세포에서 HO-1 유전자 발현의 조절과 신호전달 과정의 관계를 조사하기 위해 MAP kinase들의 특이적 저해제를 처리하고 paraquat로 자극을 준 결과, JNK 저해제인 SP600125가 가장 현저하게 paraquat에 의한 HO-1 발현을 억제하였다. 결론적으로, 도파민 세포에서 paraquat가 HO-1을 유도하는 데는 E2 enhancer가 중요하게 작용하고, AP-1과 JNK 경로를 통해 HO-1 발현이 조절된다는 사실을 처음으로 밝히게 되었다.

• Journal of Microbiology and Biotechnology
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• 제20권8호
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• pp.1196-1203
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• 2010

In the present work, methanethiol and dimethyldisulfide were investigated as sulfur sources for methionine synthesis in Corynebacterium glutamicum. In silico pathway analysis predicted a high methionine yield for these reduced compounds, provided that they could be utilized. Wild-type cells were able to grow on both methanethiol and dimethyldisulfide as sole sulfur sources. Isotope labeling studies with mutant strains, exhibiting targeted modification of methionine biosynthesis, gave detailed insight into the underlying pathways involved in the assimilation of methanethiol and dimethyldisulfide. Both sulfur compounds are incorporated as an entire molecule, adding the terminal S-$CH_3$ group to O-acetylhomoserine. In this reaction, methionine is directly formed. MetY (O-acetylhomoserine sulfhydrylase) was identified as the enzyme catalyzing the reaction. The deletion of metY resulted in methionine auxotrophic strains grown on methanethiol or dimethyldisulfide as sole sulfur sources. Plasmid-based overexpression of metY in the ${\Delta}$metY background restored the capacity to grow on methanethiol or dimethyldisulfide as sole sulfur sources. In vitro studies with the C. glutamicum wild type revealed a relatively low activity of MetY for methanethiol (63 mU/mg) and dimethyldisulfide (61 mU/mg). Overexpression of metY increased the in vitro activity to 1,780 mU/mg and was beneficial for methionine production, since the intracellular methionine pool was increased 2-fold in the engineered strain. This positive effect was limited by a depletion of the metY substrate O-acetylhomoserine, suggesting a need for further metabolic engineering targets towards competitive production strains.

• Applied Biological Chemistry
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• 제36권6호
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• pp.525-532
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• 1993

쌀의 주요 저장 단백질중의 하나인 알콜용해성 프롤라민의 성질을 분석하고 이들을 동정하기 위하여 유전자 database로부터 얻은 17개의 프롤라민 유전자의 염기서열을 상호비교 분석하였다. 유전자로부터 유추된 단백질 서열의 다중분석결과 프롤라민들은 계통발생적으로 type에서 type IV의 4개의 군으로 크게 분류할 수 있었다. 이러한 분류는 아미노산 서열 중간과 카복실 말단쪽의 짧은 결손에 의해 여러 형태의 아미노산 서열에 의한 것임을 알 수 있었다. 1군에서 4군까지의 군들은 meththionine과 cysteine과 같은 황을 포함하는 아미노산이 각각 1, 4, 10, 30%로 구성된 특징을 보여 주었다. 또한 각 군들은 등전점이 9.2, 8.2, 6.7, 7.4인 각군별로 매우 상이한 등전점을 나타내었다. 아울러 GC3s에 대한 유효 암호수(effective codon number, Nc)와 우선 암호수(preferred codon number)의 분석과 상관 그래프를 통해서 프롤라민 유전자들의 군별 전이 효율의 차이가 현저하여 프롤라민 생산 수준의 군별차이의 가능성을 제시하였다.