• 제목/요약/키워드: DNA recovery

검색결과 118건 처리시간 0.027초

Detection of Mitotic Centromere-Associated Kinesin (MCAK) During Cell-Cycle Progression of Human Jurkat T Cells Using Polyclonal Antibody Raised Against Its N- Terminal Region Overexpressed in E. coli

  • Jun, Do-Youn;Rue, Seok-Woo;Kim, Byung-Woo;Kim, Young-Ho
    • Journal of Microbiology and Biotechnology
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    • 제13권6호
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    • pp.912-918
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    • 2003
  • Mitotic centromere-associated kinesin (MCAK), which is a novel kinesin with a central motor domain, is believed to playa role in mitotic segregation of chromosome during the M phase of the cell cycle. In the present study, it is shown that a rabbit polyclonal antibody has been produced using the N-terminal region (187 aa) of human MCAK expressed in E. coli as the antigen. To express the N-terminal region in E. coli, the MCAK cDNA fragment encoding N-terminal 187 aa was obtained by PCR and was then inserted into the pET 3d expression vector. Molecular mass of the N-terminal region overexpressed in the presence of IPTG was 23.2 kDa on SDS-PAGE, and the protein was insoluble and mainly localized in the inclusion body that could be easily purified from the other cellular proteins. The N-terminal region was purified by electro-elution from the gel after the inclusion body was resolved on the SDS-PAGE. The antiserum obtained after tertiary immunization with the purified protein specifically recognized HsMCAK when subjected to Western blot analysis, and showed a fluctuation of the protein level during the cell cycle of human Jurkat T cells. Synchronization of the cell-cycle progression required for recovery of cells at a specific stage of the cell cycle was performed by either hydroxyurea or nocadazole, and subsequent release from each blocking at 2, 4, and 7 h. Northern and Western analyses revealed that both mRNA and protein of HsMCAK reached a maximum level in the S phase and declined to a basal level in the G1 phase. These results indicate that a polyclonal antibody raised against the N-terminal region (187 aa) of HsMCAK, overexpressed in E. coli, specifically detects HsMCAK (81 kDa), and it can analyze the differential expression of HsMCAK protein during the cell cycle.

Mongolian gerbil의 뇌허혈에 대한 현삼의 신경보호효과 (Neuroprotective Effects of Scrophulariae Radix on Cerebral Ischemia in Mongolian Gerbils)

  • 이준환;송미연;이종수;김성수;신현대;정석희
    • 한방재활의학과학회지
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    • 제18권4호
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    • pp.1-11
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    • 2008
  • 목적 : 뇌허혈은 일시적 혹은, 영구적 뇌동맥 폐색에 의한 뇌혈류의 감소로 유발되며, 허혈 부위에서는 복잡한 병태 생리적 과정을 통하여 신경 세포사가 초래되어 비가역적인 신경학적 손상을 일으킨다. 본 연구에서는 모래쥐를 대상으로 일시적인 전뇌허혈을 유발 시킨 후 해마 치상회에서 허혈로 인한 세포사멸을 관찰하고, 현삼(玄蔘)의 투여가 허혈로 유발된 해마 치상회에서 세포사멸에 미치는 영향과 단기 기억에 미치는 효과를 규명하고자 실험하였다. 연구방법 : 세포사멸은 DNA 분절을 나타내는 terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) 염색법과 단백분해 과정의 마지막 단계에 발현되는 caspase-3에 대한 면역조직화학법을 이용하였고, 단기기억은 step-down avoidance task를 실시하여 평가하였다. 결과 : 본 실험의 결과 일시적인 전뇌허혈은 해마 치상회의 세포사멸을 유의하게 증가시켰으며 단기기억을 감소시켰다. 현삼의 투여는 허혈로 증가된 해마 치상회의 세포사멸을 유의하게 억제하였고, 허혈로 인한 단기 기억의 감소를 유의하게 억제시켰다. 결론 : 본 실험을 통하여 현삼은 뇌허혈로 증가된 세포사멸을 억제하고 단기 기억을 향상시킴을 알 수 있었고, 따라서 현삼은 뇌허혈로 인한 뇌손상을 보호할 수 있는 효과가 있음을 제시하는 바이다.

Characterization of a Novel DWD Protein that Participates in Heat Stress Response in Arabidopsis

  • Kim, Soon-Hee;Lee, Joon-Hyun;Seo, Kyoung-In;Ryu, Boyeong;Sung, Yongju;Chung, Taijoon;Deng, Xing Wang;Lee, Jae-Hoon
    • Molecules and Cells
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    • 제37권11호
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    • pp.833-840
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    • 2014
  • Cullin4-RING ubiquitin ligase (CRL4) is a family of multi-subunit E3 ligases. To investigate the possible involvement of CRL4 in heat stress response, we screened T-DNA insertion mutants of putative CRL4 substrate receptors that exhibited altered patterns in response to heat stress. One of the mutants exhibited heat stress tolerance and was named heat stress tolerant DWD1 (htd1). Introduction of HTD1 gene into htd1-1 led to recovery of heat sensitivity to the wild type level, confirming that the decrease of HTD1 transcripts resulted in heat tolerance. Therefore, HTD1 plays a negative role in thermotolerance in Arabidopsis. Additionally, HTD1 directly interacted with DDB1a in yeast two-hybrid assays and associated with DDB1b in vivo, supporting that it could be a part of a CRL4 complex. Various heat-inducible genes such as HSP14.7, HSP21, At2g03020 and WRKY28 were hyper-induced in htd1-1, indicating that HTD1 could function as a negative regulator for the expression of such genes and that these genes might contribute to thermotolerance of htd1-1, at least in part. HTD1 was associated with HSP90-1, a crucial regulator of thermotolerance, in vivo, even though the decrease of HTD1 did not affect the accumulation pattern of HSP90-1 in Arabidopsis. These findings indicate that a negative role of HTD1 in thermotolerance might be achieved through its association with HSP90-1, possibly by disturbing the action of HSP90-1, not by the degradation of HSP90-1. This study will serve as an important step toward understanding of the functional connection between CRL4-mediated processes and plant heat stress signaling.

Cardiac Ankyrin Repeat Protein의 과량발현이 혈관내피세포에서 갖는 혈관신생 촉진 효과 (Angiogenic Effect of Cardiac Ankyrin Repeat Protein Overexpression in Vascular Endo-thelial Cell)

  • 공훈영;변종회
    • 미생물학회지
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    • 제44권4호
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    • pp.282-288
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    • 2008
  • 혈관이 좁아지거나 막혀서 생기는 허혈은 심장, 뇌, 다리와 같은 인체의 여러 장기에 영향을 미친다. 최근 혈관신생 분야에서 많은 진전이 있어 기존 치료법으로 치료가 되지 않는 허혈성 환자들의 치료 가능성에 대한 기대가 많아졌다. 혈관형성은 여러 개의 인자들과 세포들이 관여하는 복잡한 과정이기 때문에, 한 개의 인자보다는 여러 인자들을 병합하는 요법이 점점 많이 시도되고 있는데, 이런 병합요법의 한 예로 전사인자를 전달하는 전략을 생각할 수 있다. 이에 본 연구에서는 cardiac ankyrin repeat protein (CARP)의 유전자를 대상으로 그 혈관신생 능력을 혈관내피세포에서 조사하였다. 아데노바이러스 벡터 내에 human CARP의 cDNA를 클로닝하여 재조합 아데노바이러스를 제조하였으며, 이를 이용한 유전자 전달실험 결과, CARP 유전자 전달 군에서 유의하게 혈관내피세포의 중식과 모세관 구조 형성, 그리고 vascular endothelial growth factor의 발현 등을 증가시킴을 확인하였다. 본 연구 결과는 CARP가 혈관신생 연구의 새로운 목표 유전자로서 그 기능에 대한 많은 연구가 필요함을 뒷받침해준다.

Ginsenoside Rg1 supplementation clears senescence-associated β-galactosidase in exercising human skeletal muscle

  • Wu, Jinfu;Saovieng, Suchada;Cheng, I-Shiung;Liu, Tiemin;Hong, Shangyu;Lin, Chang-Yu;Su, I-Chen;Huang, Chih-Yang;Kuo, Chia-Hua
    • Journal of Ginseng Research
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    • 제43권4호
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    • pp.580-588
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    • 2019
  • Background: Ginsenoside Rg1 has been shown to clear senescence-associated beta-galactosidase (SA-${\beta}$-gal) in cultured cells. It remains unknown whether Rg1 can influence SA-${\beta}$-gal in exercising human skeletal muscle. Methods: To examine SA-${\beta}$-gal change, 12 young men (age $21{\pm}0.2years$) were enrolled in a randomized double-blind placebo controlled crossover study, under two occasions: placebo (PLA) and Rg1 (5 mg) supplementations 1 h prior to a high-intensity cycling (70% $VO_{2max}$). Muscle samples were collected by multiple biopsies before and after cycling exercise (0 h and 3 h). To avoid potential effect of muscle biopsy on performance assessment, cycling time to exhaustion test (80% $VO_{2max}$) was conducted on another 12 participants (age $23{\pm}0.5years$) with the same experimental design. Results: No changes of SA-${\beta}$-gal were observed after cycling in the PLA trial. On the contrary, nine of the 12 participants showed complete elimination of SA-${\beta}$-gal in exercised muscle after cycling in the Rg1 trial (p < 0.05). Increases in apoptotic DNA fragmentation (PLA: +87% vs. Rg1: +133%, p < 0.05) and $CD68^+$ (PLA:+78% vs. Rg1:+121%, p = 0.17) occurred immediately after cycling in both trials. During the 3-h recovery, reverses in apoptotic nuclei content (PLA:+5% vs. Rg1 -32%, p < 0.01) and increases in inducible nitrate oxide synthase and interleukin 6 mRNA levels of exercised muscle were observed only in the Rg1 trial (p < 0.01). Conclusion: Rg1 supplementation effectively eliminates senescent cells in exercising human skeletal muscle and improves high-intensity endurance performance.

백서 설신경 압박손상모델에서 신경성장인자 유전자 주입이 신경재생에 미치는 영향 (EFFECT OF NERVE GROWTH FACTOR GENE INJECTION ON THE NERVE REGENERATION IN RAT LINGUAL NERVE CRUSH-INJURY MODEL)

  • 고은봉;정헌종;안강민;김성민;김윤희;장정원;이종호
    • Maxillofacial Plastic and Reconstructive Surgery
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    • 제28권5호
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    • pp.375-395
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    • 2006
  • Purpose: Lingual nerve (LN) damage may be caused by either tumor resection or injury such as wisdom tooth extraction, Although autologous nerve graft is sometimes used to repair the damaged nerve, it has the disadvantage of necessity of another operation for nerve harvesting. Moreover, the results of nerve grafting is not satisfactory. The nerve growth factor (NGF) is well-known to play a critical role in peripheral nerve regeneration and its local delivery to the injured nerve has been continuously tried to enhance nerve regeneration. However, its application has limitations like repeated administration due to short half life of 30 minutes and an in vivo delivery model must allow for direct and local delivery. The aim of this study was to construct a well-functioning $rhNGF-{\beta}$ adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with enhanced and extended secretion of hNGF from the injured nerve by injecting $rhNGF-{\beta}$ gene directly into crush-injured LN in rat model. Materials and Methods: $hNGF-{\beta}$ gene was prepared from fetal brain cDNA library and cloned into E1/E3 deleted adenoviral vector which contains green fluorescence protein (GFP) gene as a reporter. After large scale production and purification of $rhNGF-{\beta}$ adenovirus, transfection efficiency and its expression at various cells (primary cultured Schwann cells, HEK293 cells, Schwann cell lines, NIH3T3 and CRH cells) were evaluated by fluorescent microscopy, RT-PCR, ELISA, immunocytochemistry. Furthermore, the function of rhNGF-beta, which was secreted from various cells infected with $rhNGF-{\beta}$ adenovirus, was evaluated using neuritogenesis of PC-12 cells. For in vivo evaluation of efficacy of $rhNGF-{\beta}$ adenovirus, the LNs of 8-week old rats were exposed and crush-injured with a small hemostat for 10 seconds. After the injury, $rhNGF-{\beta}$ adenovirus($2{\mu}l,\;1.5{\times}10^{11}pfu$) or saline was administered into the crushed site in the experimental (n=24) and the control group (n=24), respectively. Sham operation of another group of rats (n=9) was performed without administration of either saline or adenovirus. The taste recovery and the change of fungiform papilla were studied at 1, 2, 3 and 4 weeks. Each of the 6 animals was tested with different solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) by two-bottle test paradigm and the number of papilla was counted using SEM picture of tongue dorsum. LN was explored at the same interval as taste study and evaluated electro-physiologically (peak voltage and nerve conduction velocity) and histomorphometrically (axon count, myelin thickness). Results: The recombinant adenovirus vector carrying $rhNGF-{\beta}$ was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. GFP expression was observed in 90% of $rhNGF-{\beta}$ adenovirus infected cells compared with uninfected cells. Total mRNA isolated from $rhNGF-{\beta}$ adenovirus infected cells showed strong RT-PCR band, however uninfected or LacZ recombinant adenovirus infected cells did not. NGF quantification by ELISA showed a maximal release of $18865.4{\pm}310.9pg/ml$ NGF at the 4th day and stably continued till 14 days by $rhNGF-{\beta}$ adenovirus infected Schwann cells. PC-12 cells exposed to media with $rhNGF-{\beta}$ adenovirus infected Schwann cell revealed at the same level of neurite-extension as the commercial NGF did. $rhNGF-{\beta}$ adenovirus injected experimental groups in comparison to the control group exhibited different taste preference ratio. Salty, sweet and sour taste preference ratio were significantly different after 2 weeks from the beginning of the experiment, which were similar to the sham group, but not to the control group.

Basic Fibroblast Growth Factor (bFGF)의 방사선보호작용에 대한 실험적 연구 (In vivo Radioprotective Effects of Basic Fibroblast Growth Factor in C3H Mice)

  • 김연실;윤세철
    • Radiation Oncology Journal
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    • 제20권3호
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    • pp.253-263
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    • 2002
  • 목적 : bFGF (basic fibroblast growth factor)는 섬유아세포(fibroblast)에서 분비하는 대표적인 성장인자로 섬유아세포뿐 아니라 간질조직과 골수 및 다른 상피 근원세포의 성장에도 관여하며 방사선보호제 역할에 관한 연구가 시도되고 있다. 이 연구는 방사선보호제로서의 bFGF의 기능을 알아보고자 하였다. 대상 및 방법 : 간엽조직 기원(mesenchymal origin)인 마우스육종 180 종양세포를 생쥐 대퇴부 피하에 이식하고 bFGF를 투여한 후 전신방사선조사(6, 8, 10 Gy)하여 생쥐의 생존률을 조사하고 bFGF (3, $6\;{\mu}g$/쥐)의 방사선보호효과를 관찰하였다. 동시에 이식한 마우스 180 고형종양을 국소방사선조사한 후 bFGF가 종양성장에 미치는 영향을 알아보았다. 또한 bFGF에 의한 방사선보호효과의 기전을 이해 하고자 소장점막, 골수, 폐조직 및 이식종양조직에 대한 병리 조직학적 검사와 DNA terminal transferase nick-end labeling assay 방법으로 아포프토시스(apoptosis) 빈도를 측정하였다. 결과 : 1) 방사선조사단독군에 비해 방사선조사와 $6\;{\mu}g$ bFGF 투여병행군에서 생쥐의 골수치사를 감소시켜 생존률이 증가되었다(p<0.05). 2) 방사선조사단독군에 비해 방사선조사와 $6\;{\mu}g$ bFGF 투여병행군에서 공장 소낭선 깊이 및 미세융모 길이가 의의 있게 증가되었다(p<0.05). 소낭선세포의 아포프토시스 빈도는 방사선조사단독군에 비해 방사선조사와 bFGF 투여병행군에서 방사선조사후 8시간, 24시간에 감소하였으며 bFGF를 고용량 투여한 군에서 뚜렷하였다. 3) 골수조직에서는 방사선조사 후 7일, 14일째 세포 밀도가 방사선조사단독군에 비해 방사선조사와 $6\;{\mu}g$ bFGF 투여병행군에서 증가하였으며 특히 거핵구(megakaryocyte) 계열의 증가가 뚜렷하였다. 4) 폐조직의 H-E 염색 조직소견에서 방사선단독군과 방사선조사와 bFGF 투여병행군 간의 차이는 없었다. 5) 골수 및 폐 조직에서 bFGF 투여에 따른 초기 아포프토시스 빈도의 차이는 려었다(p>0.05). 6) 양성대조군과 bFGF단독투여군 비교시 bFGF투여에 의한 종양성장은 관찰되지 않았으며(p>0.05) 방사선조사단독군과 방사선조사와 $6\;{\mu}g$ bFGF 투여병행군에서도 종양성장곡선의 차이는 없었다(p>0.05). 결론 : 이상의 결과로 bFGF는 소장점막 및 골수세포에 방사선보호효과가 있었으며 그 기전은 조혈모세포 및 소장낭선세포의 성장 및 재생을 촉진하고 조기에 방사선으로 유도된 아포프토시스를 감소시키기 때문인 것으로 생각된다.

활락효영단합인삼양위탕(活絡效靈丹合人蔘養胃湯)이 혈관신생(血管新生) 억제(抑制)에 미치는 영향(影響) (Effect of Whalakyuoleyng-dan plus Yinsamyangwui-tang on Anti-angionesis)

  • 고기완;박준혁;강희;김성훈;유영법;심범상;최승훈;안규석
    • 대한한방종양학회지
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    • 제7권1호
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    • pp.77-97
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    • 2001
  • Anti-angiogenesis is one of therapies which have been high-lightened on the research of cancer treatment. Anti-angiogenesis means that new blood vessels are created from a existing capillary tube and it is a important process on metastasis and permeation when cancer is created or formed. Since angiogenesis have been under research, a complete recovery oriented treatment against cancer have been suggested blocking metastasis, delaying the growth of cancer cell, and blocking the supply of oxygen and nutritive substance through the web of blood vessels. Until now, there are several anti-angiogenesis, which have been known to the public, such as thalidomide, angiostatin, endostatin, 2-methoxyestradiol, TNP-470, and marimastat, etc. Additionally, 17 clinical testing projects about anti-angiogenesis are on the process in NCI(National Cancer Institute). Especially, TNP-470 showed effectiveness against cancer on clinical testing after finishing animal testing. Based on existing researches showing that Yinsamyangwui-tang is effective to strengthening body resistance and Whallakhyolenyng-dan effects cells on the inside of blood vessel because Whallakhyolenyng- dan restrains cell adhesion during the restraining period of a blood vessel, I tried to research the effect of Whalakhyolenyng-dan plus Yinsamyangwui-tang on angiogenesis. I made a conclusion putting into operation through using SK-Hep-1 (KCLB 30052), A549(KCLB 10185), AGS(KCLB 21739), and BCE(Bovine Capillary Endothelial Cell). Followings are the results of my experimental research: 1. According to the researching results of anti-cancer activation against cancer cell, Whallkhyoleyng dan plus Yinsamyangwui-tang decreased the number of cancer cells -- While injecting $600{\mu}g/ml$, injected groups decreased 3.1% more comparing with the contrastive group of SK-Hep-1, 49.7% more comparing with the contrastive group of A549, and 31.0% more comparing with the contrastive group of AGS. 2. According to the researching results of DNA composition effect between BCE and cancer cell, Whallakhyoleyng-dan plus Yinsamyangwui-tang reduced the rate of SK-Hep-1 synthesis inhibition by 59.1% at $600{\mu}g/ml$ intensity comparing with contrastive group; for A549, 72.6%; for AGS, 6.1%, for BCE, 28.9%. 3. According to the researching results about the effect of BCE cell to angiogenesis, angiogenesis was restrained at $400{\mu}g/ml$ intensity during 18 hours observation. 4. In the case of aortic ring assay, the half level of angiogenesis was reduced comparing with the contrastive group while injecting with $400{\mu}g/ml$ intensity; with $800{\mu}g/ml$, under 10% comparing with contrastive group; and with $1600{\mu}g/ml$, complete restrain. According to the above results, Whallakhyoleyng-dan plus Yinsamyangwui-tang was proved to have an anti-angiogenetic effects.

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