• 혜화의학회지
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• 제10권2호
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• pp.179-188
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• 2002

This experimental study was carried out to evaluate the effects of Acanthopanacis cortex on Cytokine-inducing and and immune response in Mice. In order to investigate the effect of Acanthopanacis cortex, the following was performed; Cytotoxicity, in vitro, the fraction of $CD4^+$, $CD8^+$, $B220^+$ in splenic cell, gene expression of IL-12(p35), IL-12(p40), IFN-${\gamma}$, and splenic cell proliferation by Acanthopanacis cortex. Analysis of cytokine gene expression was carried out by RT-PCR amplification. Amplified PCR products were electrophoresed on 1.2% agarose gel, and the analysis (Ht) was used to 1D-density program. The results were obtained as follows. Acanthpanacis cortex showed didn't have cell toxicity under $12{\mu}g/m{\ell}$ group on mouse lung fibroblast cells. In an in vitro model using mouse peripheral blood mononuclear cells (PBMCs), extract of Acanthpanacis cortex induced multiple cytokine, including interleukin-12 (p35), interleukin-12 (p40), interferon-gamma (IFN-${\gamma}$). The extract also enhanced the percentages of the $CD4^+$, and $CD8^+$ in the untreated control were $22.1{\pm}3.3$ to $38.4{\pm}2.1$, and $5.0{\pm}0.4$ to $10.7{\pm}0.3%$, respectively. From above findings, it is suggested that Acanthopanacis cortex is able to anti-cancer and activate immune response system.

• Journal of Microbiology
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• 제45권2호
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권5호
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• pp.389-392
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• 2004

In order to produce a harmful protein more efficiently, this expression cassette, dubbed pCol-MICT, is directed by the colicin promoter, and was constructed by the insertion of a $rrnBT_1T_2$ fragment of pEXP7, and a MxelnteinCBD fragment of pTXB3, into pSH375. To test whether harmful proteins, including proteolytic enzymes, could be effectively produced by this cassette, the carboxypeptidase (CPase) Taq gene was inserted into the pCol-MICT cassette to yield pCol-CPase Taq-MICT. E coli W3l 10 tells harboring pCol-CPase Taq-MICT produced a large quantity of this enzyme, as much as 47.2 mg of purified from per liter of culture, when cultured in the presence of mitomycin C ($0.4{\mu}g/mL$). This indicates that the colicin promoter-controlled E, coli expression cassette was able to produce almost 8 times of protein than the conventional tar promoter-based system, and that this cassette may be useful in the Synthesis of other harmful proteins.

• Archives of Pharmacal Research
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• 제21권3호
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• pp.291-297
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• 1998

Cop protein has been overexpressed in Escherichia coli using a T7 RNA polymerase system. Purification to apparent homogeneity was achieved by the sequential chromatography on ion exchange, affinity chromatography, and reverse phase high performance liquid chromatography system. The molecular weight of the purified Cop was estimated as 6.1 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). But the molecular mass of the native state Cop was shown to be 19 kDa by an analytical high performance size exclusion chromatography, suggesting a trimer-like structure in 50 mM Tris-HCI buffer (pH 7.5) containing 100 mM NaCl. Cop protein Was calculated to contain $39.1% {\alpha}-helix, 16.8% {\beta}-sheet$, 17.4% turn, and 26.8% random structure. The DNA binding property of Cop protein expressed in E. coli Was preserved during the expression and purification process. The isoelectric point of Cop was determined to be 9.0. The results of amino acid composition analysis and N-terminal amino acid sequencing of Cop showed that it has the same amino acid composition and N-terminal amino acid sequence as those deduced from its DNA sequence analysis, except for the partial removal of N-terminal methionine residue by methionyl-aminopeptidase in E. coli.

• 대한한방내과학회지
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• 제31권2호
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• pp.242-253
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• 2010

Objectives : The purpose of this study was to examine the anti-inflammatory effects Forsythiae Fructus on LPS-activated RAW264.7 cells. Methods : Statistical significance was examined through measuring MTT, nitric oxide (NO), TNF $\alpha$, IL 6, NOS2 and COX2 of LPS-activated RAW264.7 cells. Results : In the toxicity experiment of FFE, NO significant toxicity was shown on cells in the concentration ranges of 100, 200 and 300 ${\mu}g/ml$. FFE dose dependently decreases 5.49, 25.41, 33.64 % in LPS induced NO production (p<0.05, p<0.001 and p<0.001), however, FFE don't show significant difference in LPS-induced TNF $\alpha$ and IL 6. Furthermore, FFE showed inhibiting tendency against the revelation of NOS2 and COX2 in LPS-activated RAW264.7 cells, dose-dependently according to concentration. Conclusions : These results mean that FFE is effective for anti-inflammation via inhibition of NOS2 and COX2 expression.

• 한국미생물·생명공학회지
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• 제24권4호
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• pp.415-422
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• 1996

A gene coding for a pyruvyl transferase enzyme involved in exopolysaccharide biosynthesis of Zoogloea ramigera 115SLR was isolated and sequenced. A 4.5 kb of BamHI DNA fragment was isolated from chromosomal DNA using a probe derived from ketal pyruvyl transferase gene of Xanthomonas campestris. The nucleotide sequence of 2.66 kb Pst1/HindIII DNA fragment which was homology with a probe revealed the existence of two complete open reading frames (ORF2 and ORF3) and two partial open reading frames (ORFI and ORF4). The deduced amino acid sequence of ORF3 was homologous to the ketalase (GumL product) of X campestris with 49.5% of similarity and 21.6% of identity. ORF2 on the other hand showed the higher identity with the ketalase (ExoV product) of Rhizobium meliloti (36%) as well as the ketalase of X campestris (23%) than that of ORF3. A gene product of ORF2 was determined with a bacteriophage T7 RNA polymerase/promoter system in E. coli. The molecular weight of protein was 33,500 dalton.

• 생명과학회지
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• 제28권9호
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• pp.1030-1041
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• 2018

환경 스트레스는 식물의 성장을 저해하며 작물의 생산량을 감소시키는 주요 원인이다. 식물은 다양한 유전자의 발현 변화를 통해 스트레스에 대한 저항성을 나타낸다. 본 연구에서는 activation tagging system을 이용하여 기존에 밝혀지지 않은 새로운 고염 스트레스 반응 유전자들을 분리하였다. 애기장대의 발아 단계에서 고염 스트레스에 저항성을 보이는 9개의 activation tagging 라인을 선별하였다. 그 중 TAIL-PCR 방법을 이용하여 AT7508, AT7512, AT7527, AT7544, AT7548, AT7556의 6개 라인에서 T-DNA가 삽입된 위치를 확인하였으며 각 라인에서 T-DNA가 삽입된 주변 유전자의 발현을 RT-PCR로 분석하였는데 AT7508, AT7512, AT7527, AT7544, AT7556에서 각각 ClpC2/HSP93-III (At3g48870), plant thionin family (At2g20605), anti-muellerian hormone type-2 receptor (At3g50685), vacuolar iron transporter family protein (At4g27870), microtubule-associated protein (At5g16730)이 activation 된 것으로 밝혀졌다. 더불어 AT7548에서는 T-DNA가 삽입된 곳의 양쪽에 위치하는 두 유전자인 Arabinogalactan protein 13 (AGP13) (At4g26320)과 F-box/RNI-like/FBD-like domains-containing protein (At4g26340)이 모두 activation 되었다. Activation 된 7개 유전자는 기존에 고염 스트레스 저항성과 관련된 기능이 알려지지 않은 유전자로 본 연구를 통해 새롭게 고염 스트레스 반응에 대한 기능이 밝혀졌다. 7개의 activation된 유전자 중 ClpC2/HSP93-III, AGP13, F-box/RNI-like/FBD-like domains-containing protein의 3개 유전자는 고염 스트레스에 의해 발현이 증가하였다. 또한 AT7508과 AT7527, AT7544 라인은 발아 단계뿐만 아니라 유식물체 발달 과정에서도 고염 스트레스 저항성을 보여 activation tagging 라인의 선별 결과의 타당성을 뒷받침 하였다. 본 연구의 결과를 통해 activation tagging system이 새로운 스트레스 반응 유전자를 찾아낼 수 있는 유용한 기술임을 확인할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제31권8호
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• pp.1079-1087
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• 2021

Gentisic acid (GA), a benzoic acid derivative present in various food ingredients, has been shown to have diverse pharmaceutical activities such as anti-carcinogenic, antioxidant, and hepatoprotective effects. In this study, we used a co-culture system to investigate the mechanisms of the anti-inflammatory and anti-adipogenic effects of GA on macrophages and adipocytes, respectively, as well as its effect on obesity-related chronic inflammation. We found that GA effectively suppressed lipopolysaccharide-stimulated inflammatory responses by controlling the production of nitric oxide and pro-inflammatory cytokines and modulating inflammation-related protein pathways. GA treatment also inhibited lipid accumulation in adipocytes by modulating the expression of major adipogenic transcription factors and their upstream protein pathways. Furthermore, in the macrophage-adipocyte co-culture system, GA decreased the production of obesity-related cytokines. These results indicate that GA possesses effective anti-inflammatory and anti-adipogenic activities and may be used in developing treatments for the management of obesity-related chronic inflammatory diseases.

• Asian-Australasian Journal of Animal Sciences
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• 제21권7호
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• pp.980-987
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• 2008

The objective of this study was to investigate the effects of cell status of BOEC on development of bovine IVM/IVF embryos and gene expression in BOEC before or after culturing of embryos. The developmental rates beyond morula stage in the BOEC co-culture group was significantly higher than in the control group (p<0.05). In particular, blastocyst production in the BOEC co-culture group (28.3%) was dramatically increased compared with the control group (7.2%). In the in vitro development of bovine IVM/IVF embryos according to cell status, the developmental rates beyond morula stage in the primary culture cell (PCC) co-culture group were the highest of all experimental groups. Expression of genes related to growth (TGF-${\beta}$ EGF and IGFBP), apoptosis (Bax, Caspase-3 and p53) and antioxidation (CuZnSOD, MnSOD, Catalase and GPx) in different status cells of BOEC for embryo culture was detected by RT-PCR. While EGF gene was detected in isolated fresh cells (IFC) and PCC, TGF-${\beta}$ and IGFBP were found in IFC or PCC after use in the embryo culture, respectively. Caspase-3 and Bax genes were detected in all experimental groups regardless of whether the BOEC was used or not used in the embryo culture. However, p53 gene was found in IFC of both conditions for embryo culture and in frozen/thawed culture cells (FPCC) after use in the embryo culture. Although antioxidant genes examined were detected in all experimental groups before using for the embryo culture, these genes were not detected after use. This study indicated that the BOEC co-culture system used for in vitro culture of bovine IVF embryos can increase the developmental rates, and cell generations and status of BOEC might affect the in vitro development of bovine embryos. The BOEC monolayer used in the embryo culture did not express the growth factors (TGF-${\beta}$ and EGF) and enzymatic antioxidant genes, thereby improving embryo development in vitro.

• 한국해양생명과학회지
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• 제7권1호
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• pp.15-20
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• 2022

본 연구는 우리나라 해안에서 널리 서식 중인 해양 자원 중 하나인 전복(Haliotis discus hannai)의 차세대염기서열분석 데이터 기반으로 선별한 신규 펩타이드의 항암 활성을 평가한 연구이다. 펩타이드의 항암 활성은 교모세포종 세포주인 SNU-489에서 농도 의존적으로 처리 시간에 비례하여 증가하였으며, 200 µM로 48시간 처리하였을 때 암 세포 사멸율이 67%로 가장 높게 나타났다. 반면 정상 세포인 HaCaT에서 가장 높은 세포 사멸율은 18%로 농도 의존적이었으나 처리 시간과는 무관하였다. 또한 신규 펩타이드의 항암 메커니즘 과정을 밝히기 위해 세포자멸괴사(Necroptosis) 관련 유전자의 발현 변화를 qRT-PCR 방법을 통해 검증하였다. RIPK3는 신규 펩타이드 처리군에서 200 µM 처리 시 9배 이상 발현 증가, MLKL는 100 µM 처리군에서 대조군 대비 2배 이상 유의미하게 발현이 증가되었다. 이러한 결과로 미루어 볼 때, 전복 유래 신규 펩타이드는 암 세포 특이적으로 세포 독성을 가지며, 세포자멸괴사 메커니즘을 통해 암세포 사멸을 일으키는 것으로 추측되므로 신규 펩타이드가 추후 교모세포종 치료제의 후보 물질로 활용될 수 있을 것으로 사료된다.