• Title/Summary/Keyword: PCR inhibitor

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자궁내막증 환자와 정상 여성의 자궁내막에서 TIMP-3와 PAI-1 mRNA 발현 차이에 관한 연구 (Endometrium from Women with Endometriosis Expresses Decreased Levels of Plasminogen Activator Inhibitor-1 and Tissue Inhibitor of Metalloproteinase-3 Compared to Normal Endometrium)

  • 정혜원
    • 한국발생생물학회지:발생과생식
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    • 제3권1호
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    • pp.29-38
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    • 1999
  • 자궁내막증은 흔한 부인과적 질병이며 여성 불임의 한 원인이 되나 그 발생 원인에 대하여서는 아직 논란의 여지가 많다. 최근 월경혈의 역류가 한 원인이며 자궁내막증 환자가 정상여성에서 보다 역류되는 월경혈의 양이 많거나 침습성이 강한 것이 자궁내막증의 발생 원인이 될 수 있다는 이론들이 소개되었다. 종양이나 자궁내막 조직의 침습이나 전이에는 세포막외 기질 및 기저막의 파괴가 일어나야 하는데 이 과정에 plasminogen activators (PAs)나 matrix metalloproteinase (MMPs)같은 proteolytic enzyme이 관여한다. 이에 자궁 내막증환자와 대조군의 자궁내막에서 PA나 MMP를 억제하는 plasminogen activator inhibitor-1 (PAI-1)나 tissue inhibitor of metalloproteinase (TIMP-3)의 mRNA 발현의 차이를 quantitative competitive RT PCR로 연구하였으며, 그 결과 자궁내막증 환자의 황체기 자궁내막에서는 정상 대조군 환자에서 보다 PAI-1과 TIMP-3 mRNA발현이 낮음을 알 수 있었다. 따라서 자궁내막증 환자의 자궁내막에서는 PA와 MMP의 활성도가 증가할 수 있으며 이 증가된 proteolytic activity로 인하여 역류된 자궁내 막 조직의 복강내 침습이 보다 쉽게 일어날 가능성이 있다.

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Effect of Endoplasmic Reticulum (ER) Stress Inhibitor Treatment during Parthenogenetic Activation on the Apoptosis and In Vitro Development of Parthenogenetic Porcine Embryos

  • Park, Hye-Bin;Kim, Mi-Jeong;Jung, Bae-Dong;Lee, Seunghyung;Park, Choon-Keun;Yang, Boo-Keun;Cheong, Hee-Tae
    • 한국발생생물학회지:발생과생식
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    • 제22권3호
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    • pp.235-244
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    • 2018
  • We investigate the effect of endoplasmic reticulum (ER) stress inhibitor treatment during parthenogenetic activation of oocytes on the ER stress generation, apoptosis, and in vitro development of parthenogenetic porcine embryos. Porcine in vitro matured oocytes were activated by 1) electric stimulus (E) or 2) $E+10{\mu}M$ Ca-ionophore (A23187) treatment (EC). Oocytes were then treated by ER stress inhibitors such as salubrinal (200 nM) and tauroursodeoxychloic acid (TUDCA, $100{\mu}M$) for 3 h prior to in vitro culture. Parthenogenetic embryos were sampled to analyze ER stress and apoptosis at the 1-cell and blastocyst stages. The x-box binding protein 1 (Xbp1) mRNA and ER stress-associated genes were analyzed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. At the 1-cell stage, although no difference was observed in Xbp1 splicing among treatments, BiP transcription level in the E group was significantly reduced by salubrinal treatment, and GRP94 and ATF4 transcription levels in EC group were significantly reduced by all treatments (p<0.05) compared to control. In the EC group, both apoptotic genes were reduced by ER stress inhibitor treatments compared to control (p<0.05) except Caspase-3 gene by TUDCA treatment. These results suggest that the treatment of ER stress inhibitor during parthenogenetic activation can reduce ER stress, and thereby reduce apoptosis and promote in vitro development of porcine parthenogenetic embryos.

꽃양배추로의 Proteinase Inhibitor II ( PI-II ) 유전자 도입 (The Introduction of Proteinase Inhibitor II (PI-II) Gene into Flowering Cabbage, Brassica oleracea var. acephala DC.)

  • 김창길;정재동;안진흥
    • 식물조직배양학회지
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    • 제25권1호
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    • pp.45-50
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    • 1998
  • 꽃양배추의 하배축 조직을 proteinase inhibitor II 유전자가 도입된 Agrobacterium tumefaciens LBA 4404와 2일간 pH 5.5로 조절된 MS 액체배지에서 공동배양후 carbenicillin 500mg/L kanamycin 20mg/L와 BA 1mg/L가 함유된 MS 재분화배지에 옮겼다. 이들 조직을 매 2주마다 계대배양하였으며 약 4주후에 kanamycin 저항성 개체를 얻었다. 형질전환된 것으로 추정되는 식물체는 kanamycin 30mg/L가 함유된 선발배지에서 생존하였다. PCR 분석결과, PI-II 유전자가 형질전환체의 게놈상에 삽입되어 있음을 확인하였다. 형질전환체의 Southern blot 분석을 통하여 ECL-labelling된 PI-II 유전자와 동일한 것으로 판단되는 약 500bp 위치에서 밴드를 확인할 수 있었다.

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감자 단백질 분해효소 억제제-II 유전자로부터의 폴리펩타이드 카이모트립신 저해제와 homology가 있는 유전자단편의 클로닝 및 대장균에서의 발현 (Cloning of Gene Fragment having Homology with the Polypetide Chymotrypsin Inhibitor from the Potato Proteinase Inhibitor II Gene and Its Expression in E. coli.)

  • 정진;박상규
    • Applied Biological Chemistry
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    • 제38권5호
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    • pp.382-386
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    • 1995
  • 감자의 단백질 분해효소 억제제-II(PI-II)단백질은 카이모트립신 저해부위와 트립신 저해부위로 나뉘어 있는데 PI-II 유전자중의 하나인 PI-IIT 유전자의 염기서열에서 비롯된 아미노산 서열이 폴리펩타이드 카이모트립신 저해제(PCI) 단백질의 아미노산 서열과 84%의 높은 동질성을 가지고 있으므로 감자의 단백질 분해효소 억제제유전자 집단 (family) 중의 하나인 PCI와 homology가 있는 유전자단편을 클로닝하기 위하여 이미 클론된 PI-IIT 유전자로부터 PCR을 행하여 얻어진 DNA 단편을 백터에 클로닝하고 염기서열을 결정하였다. 염기서열을 확인한 유전자 단편을 박테리오파아지 T7 promoter와 terminator를 갖고있는 플라스미드 pET3a에 옮겨 대장균 BL2l(DE3)에서 발현시켰던바 IPTG의 부가에 따라 유기되는 것을 확인 하였으나 발현수준은 기대했던것에 미치지 못하였다.

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Paclitaxel Stimulates Cyclooxygenase-2 Expression via MAP Kinase Pathway in Rabbit Articular Chondrocytes

  • ;김송자
    • 대한의생명과학회지
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    • 제15권2호
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    • pp.141-146
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    • 2009
  • Paclitaxel, an antimicrotubule agent, binds to beta-tubulin in the microtubule and stabilizes the polymer, thereby repressing dynamic instability. Here, we have demonstrated that microtubule cytoskeletal architecture involved in regulation of the COX-2 expression in chondrocyte treated with paclitaxel. Paclitaxel enhanced COX-2 expression and prostaglandin E2 production, as indicated by the Western blot analysis, reverse transcriptase PCR(RT-PCR) and immunofluorescence staining, and $PGE_2$ assay, respectively. In our previous data have shown that paclitaxel treatment stimulated activation of ERK-1/2 and p38 kinase(Im et al., 2009). SB203580, an inhibitor of p38 kinase, blocked the induction of COX-2 expression by paclitaxel. Also PD98059, an inhibitor of ERK-1/2 kinase was blocked the induced COX-2 expression. These results indicate that activation of ERK-1/2 and p38 kinase is required for COX-2 expression induced by paclitaxel in rabbit articular chondrocytes.

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Mornitoring and Identification of Human Astrovirus from Groundwater in Korea Based on Highly Sensitive RT-nested PCR Primer Sets

  • Lee, Siwon;Bae, Kyung Seon;Park, Jihyun;Kim, Jin-Ho;Lee, Jin-Young;Choi, Jiwon;Park, Eung-Roh;You, Kyung-A
    • 대한의생명과학회지
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    • 제27권4호
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    • pp.255-263
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    • 2021
  • Human Astrovirus (HuAstV) is an important gastrointestinal pathogen that is frequently reported worldwide. Monitoring of contaminated groundwater has been suggested since HuAstV is transmitted through the fecal-oral route. This study developed a test method based on conventional reverse transcription (RT)-nested polymerase chain reaction (PCR) that involves SL® non-specific reaction inhibitor for unknown non-specific amplification taking place in the groundwater environment. An optimal method for detecting HuAstV in groundwater sample through analysis and comparison against conventionally reported method was also suggested. The developed method enabled the production of nested PCR amplicon of 630 nt, which is a sufficient length for similarity analysis based on sequencing and genotyping. Amplicons suspected to be HuAstV were amplified in two out of the twenty groundwater samples collected in Korea, presenting 99.77% and 99.73% similarity against HuAstV 1 strain lhar/2011/kor (JN887820.1) in sequencing, respectively. These amplicons were identified as HuAstV 1.

Development of a Virus Elution and Concentration Procedure for Detecting Norovirus in Cabbage and Lettuce

  • Moon, Aerie;Hwang, In-Gyun;Choi, Weon-Sang
    • Food Science and Biotechnology
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    • 제18권2호
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    • pp.407-412
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    • 2009
  • In this study, a rapid and efficient concentrating procedure that can be used for detecting viruses in vegetables was developed. The Sabin strain of poliovirus type 1 was used to evaluate the efficiency of virus recovery. The procedure included: (a) elution with 0.25 M threonine-0.3 M NaCl pH 9.5; (b) polyethylene glycol (PEG) 8000 precipitation; (c) chloroform extraction; (d) 2$^{nd}$ PEG precipitation; (f) RNA extraction; (g) reverse transcription-polymerase chain reaction (RT-PCR) combined with semi-nested PCR. The overall recoveries by elution/concentration were 29.0% from cabbage and 13.7% from lettuce. The whole procedure usually takes 18 hr. The overall detection sensitivity was 100 RT-PCR units of genogroup II norovirus (GII NoV)/25 g cabbage and 100 RT-PCR units of GII NoV/10 g lettuce. The virus detecting method developed in this study should facilitate the detection of low levels of NoV in cabbage and lettuce.

치수 및 치근단 질환에서의 단백분해효소 및 단백분해효소 억제제의 활성도에 관한 연구 (A STUDY ON THE ACTIVITY OF PROTEINASE AND PROTEINASE INHIBITOR IN PULPAL AND PERIAPICAL PATHOSES)

  • 김진우;;임성삼
    • Restorative Dentistry and Endodontics
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    • 제25권4호
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    • pp.509-526
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    • 2000
  • It is known that injuries to the dentin have a corresponding inflammatory effect on the pulp and these inflammatory effects frequently result in pulpal pathoses due to progressive degradation of pulpal connective tissue. It was supposed that the tissue degradation in different inflammatory process was controlled by proteinase activity and antiproteinase activity. Therefore, the purpose of this study was to examine the pulp and periapical pathoses in terms of the activities of proteinase and proteinase inhibitor, 37 pulpal tissues were divided by clinical diagnostic criteria into normal pulp, acute inflamed pulp, and chronic inflamed pulp, and then those groups were subdivided by histopathological findings into 5 pulpal pathoses groups, i.e. normal pulp (P1, n=8), chronic pulpitis with fibrotic change (P2, n=2), chronic pulpitis with dystrophic calcification (P3, n=11), chronic pulpitis with pulp abscess (P4, n=7), acute pulpitis with necrotic change (P5, n=4), 26 periapical tissues were also divided by ordinary histopathological findings into 3 periapical pathoses group, i.e., granuloma (A1, n=17), cyst (A2, n=2) and abscess (A3, n=7). The activities of proteinases (cathepsin G, MMP-3) and proteinase inhibitors (${\alpha}1$-AT, TIMP-1 and, SLPI) were evaluated by RT-PCR and immunohistochemical methods. The results were as follows. 1. Generally, the intensity of immunohistochemical staining of proteinases and proteinase inhibitors increased in P2 and P5 groups compared to P1 group. 2. The immunohistochemical stain of proteinases and proteinase inhibitors was intensely detected in P2 group, showing low inflammatory reaction and low tissue degradation, but it was reduced in P3 and P4 groups, showing severe tissue degradation. 3. The distribution of proteinases and proteinase inhibitors in pulpal pathoses was consistently presented by immunohistochemical staining, while the expression of proteinase and/or proteinase inhibitors mRNAs in pulpal pathoses was occasionally detected by RT-PCR methods. 4. RT-PCR of proteinase and proteinase inhibitors was usually positive in P2, showing rare tissue degradation, but it was almost negative in P3 and P4, showing severe tissue degradation. 5. We presume that the reason why the level of proteinase and proteinase inhibitors was so sparse in RT-PCR method is due to the abrupt decrease of mRNA synthesis or degradation of synthesized mRNA of proteinase and/or proteinase inhibitors depend on the inflammatory reaction and/or on the degradation of pulp tissues(P3, P4). 6. Pulpal pathoses groups showed significant lower RT-PCR detection of proteinases and proteinase inhibitors than the periapical pathoses group(p<0.05), and there is no significant difference among the periapical pathoses groups(p>0.05).

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THP-1 단핵구의 펩티도글리칸 유래 인터루킨-1 알파 발현에서 TLR2, PI3K/Akt/mTOR, MAPKs의 역할 (Involvement of Multiple Signaling Molecules in Peptidoglycan-induced Expression of Interleukin-1α in THP-1 Monocytes/Macrophages)

  • 허원;손용해;조혁래;김관회
    • 생명과학회지
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    • 제32권6호
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    • pp.421-429
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    • 2022
  • 본 연구에서는 죽상경화 플락에서 발견되는 펩티도글리칸이 혈관염증에서 어떠한 역할을 하는지 알아보기 위하여 염증성 사이토카인의 한 종류인 인터루킨-1 알파의 발현에 대한 영향을 조사하였다. 실험방법으로는 혈관염증을 주도하는 단핵구/대식세포인 THP-1 세포주에 펩티도글리칸을 처리하고 인터루킨-1 알파의 발현을 RT-PCR, real-time PCR, ELISA 방법으로 분석하였다. 펩티도글리칸의 처리 시간과 농도에 비례하여 단핵구/대식세포에서 인터루킨-1 알파의 전사체와 단백질 분비가 증가함을 관찰하였다. 또한 펩티도글리칸의 작용기전을 규명하기 위하여 신호전달을 차단하는 억제제를 세포에 처리하고 인터루킨-1 알파의 발현을 조사하였다. TLR2/4의 억제제인 OxPAPC 그리고 세포 kinase의 작용을 억제하는 LY294002(PI3 kinase 억제), Akti IV (Akt 억제), rapamycin (mTOR 억제), U0126 (MEK 억제), SB202190 (p38 MAPK 억제), SP6001250 (JNK 억제), DPI (NOX 억제)를 처리하는 경우 인터루킨-1 알파 전사체의 발현 그리고 단백질의 분비가 감소되었다. 반면에 LPS의 작용을 억제하는 polymyxin B는 인터루킨-1 알파의 발현에 영향을 주지 않았다. 이상의 결과는, 펩티도글리칸이 TLR2, PI3K, Akt, mTOR, MAPKs를 통하여 단핵구/대식세포의 인터루킨-1 알파 발현을 증가시키고 혈관염증에 기여한다는 것을 나타낸다.

골수기질세포에서 방사선 반응 유전자로서의 Plasminogen Activator Inhibitor-1 (Plasminogen Activator Inhibitor-1 as a Radiation-Responsive Gene in Bone Marrow Stromal Cells)

  • 송지연;권형주;박찬규;조덕연;이영희
    • 한국발생생물학회지:발생과생식
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    • 제9권1호
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    • pp.43-48
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    • 2005
  • 조혈세포의 주요 서식지가 되는 골수기질세포는 줄기세포의 운영을 결정하는 다양한 인자들을 제공한다. 방사선 요법은 항암치료법으로 널리 활용되고 있으나, 조혈세포의 파괴로 인한 부작용이 심각한 문제로서 조혈세포에 의한 혈액 세포가 빠른 시간 내에 회복되는 것이 필수적이다. 본 연구에서는 방사선을 조사했을 때의 줄기세포 서식지를 구성하는 세포인 골수기질세포에서 발현되는 유전자를 탐색하여 그 기능과 조절 및 혈액 형성을 이해하는 기초를 마련하고자 하였다. 방법론적으로는 polymerase chain reaction(PCR) 및 agarose 전기영동 방법을 활용한 differential display를 활용하였으며, 결과로서 여러 후보 유전자가 선별되었으나, plasminogen activator inhibitor-1(PAI-1) 유전자만이 감마선에 의해 유도됨이 반복 확인되었다. PAI-1 유전자 유도의 의미는 향후에 더 연구해야 할 것이다.

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