우리는 미카엘라 데 마르티노, 윌프레드 R. 하겐, 유용한 토론 Kourosh Honarmand Ebrahimi에 감사합니다. 이 작품은 기술의 델프트 대학에서 기금을 시작으로 지원되었다.

1. DPS 발현과 정제 높은 순도의 단백질을 얻기 DNA 보호 분석을위한 필수적인 첫 번째 단계입니다. DPS 단백질의 정제는 4 5 일에서 수행 할 수 있습니다. <ol><li> E.의 단백질 분해 효소 결핍 긴장을 변환 DPS 단백질 인코딩 시퀀스가 복제 된 어떤에 애완 동물 벡터 (예 : pET17 등)와 대장균 (예 : BL21 (DE3) pLysS 등). </li><li> 킬 적절한 항생제 (예 : 1.1에서 제공하는 예제 암피실린 및 클로람페니콜 등)를 포함 루리아 국물 (LB) 한천 플레이트에 세포를 변형. 37에서 하룻밤 번호판을 품어 ° C. </li><li> 적절한 항생제를 포함하는 LB 매체의 30 ML에 플레이트에서 단일 콜로니를 접종한다. 37 밤새 품어 ° C 200 ~ 250 rpm으로 흔들어. </li><li> 10 ML 야간 문화의 각각 적절한 항생제를 포함하는 2 x 1 L의 LB 배지에 접종한다. OD 600에 떨고 동안 37 ° C에서 성장 </서브&gt; 약 0.6. (눈에 띄게 최종 제품의 순도를 손상시키지 않고, 더 높은 OD 증가 단백질 수율에 세포 성장을.) 0.3 mM의 농도로 IPTG 첨가하여 공격력의 발현을 유도하고, 흔들어 섞으면 서 37 ° C 3 ~ 4 시간 동안 배양한다. </li><li> 15 분 6,000 XG에서 원심 분리하여 수확 세포. 유도 세포 배양의 L 당 DEAE 버퍼 (50 MM의 HEPES - 코이, 산도 7.5, 100 MM의 염화나트륨, 0.1 mM의 EDTA) 7.5 ㎖에 세포 펠렛을 resuspend을. 샘플은 액체 질소에 냉동 원하는 경우, -80 ° C에 저장할 수 있습니다. 절차는 다음 4 ℃에서 물을 욕조에 샘플을 해동 계속 될 수있다 </li><li> 과발현 공격력의 저하를 방지하기 위해 세포 현탁액에 프로테아제 억제제 (예 : Calbiochem 세트 III 단백질 분해 효소 억제제로 세포 현탁액의 ML 당 0.167 μL)에서의 혼합물을 추가합니다. </li><li> 프랑스 프레스를 사용하여 세포가없는 추출물을 준비합니다. 20 ML DEAE 버퍼 (연속 흐름 모델을 사용하는 경우), 다음 운영 중단과 총리는 프랑스 프레스t 20 KPSI 두 번 샘플. 불용성 입자를 명확히하기 위해 4 ° C에서 35 분 동안 30,000 XG에서 해물을 원심 분리기 및 뜨는을 저장합니다. 원하는 경우 상층 액을 한 후, 액체 질소를 사용하여 냉동 및 -80 ° C에 저장할 수 있습니다. </li><li> FPLC를 사용하여 DEAE 버퍼와 함께 30 ML DEAE 세파 CL-6B 열을 평형. 배경 0.2 MPa의의 최대 압력으로 1-2 ML / 분에 열을 버퍼를 실행합니다. 칼럼에있는 셀이없는 추출물을로드하고 흐름을 통해 OD 280 신호가 기준보다 증가하기 시작 한 번 수집하기 시작합니다. DEAE 버퍼 열을 씻어 지속적으로 흐름을 통해 수집하는 동안 OD 280 값이 기준에 돌아올 때까지. 흐름을 통해 바인딩 된 DNA에서 무료로 DPS 단백질의 대부분을 포함해야합니다. 100 % 버퍼 B (50 MM의 HEPES - 코이, 산도 7.5, 1 M의 NaCl, 0.1 mM의 EDTA)로 컬럼을 씻으십시오. 이 용출액은 DPS-DNA 복합체뿐만 아니라 다른 단백질을 포함하고 삭제할 수 있습니다. </li><li> 광고를 제거황산 암모늄 침전을 통해 단백질을 추가적인 오염. 풀링 흐름을 통해의 볼륨을 결정합니다. 천천히 (20 분 동안) 4시에 62 % 포화 (솔루션의 ML 당 390 밀리그램)에 ° C 잘 교반하면서 건조한 작은 입자 황산 암모늄을 추가합니다. 완전한 평형을 보장하기 위해 황산 암모늄의 마지막 부분을 추가 한 후 추가로 20 ~ 30 분 동안 혼합물을 저어. 오염 단백질 용액 침전 할 때 DPS는, 용해 상태로 유지됩니다. </li><li> 4에서 30 분 동안 20,000 XG에서 원심 분리하여 침전 된 단백질을 제거 ° C 및 뜨는을 저장합니다. </li><li> 천천히 94 % 포화에 도달하고 완전한 평형을 보장하기 위해 20-30 분 동안 저어 상등액의 ML 당 추가로 227 mg을 황산 암모늄을 추가합니다. 공격력이 높은 염 농도에서 침전, 4 ℃에서 30 분 동안 20,000 XG에서 원심 분리하여 수집 할 수 있습니다 DPS는 펠릿에있을 것이다 상층 액을 삭제할 수 있습니다. 펠렛은 -80 ° C에 저장할 수 있습니다 경우 데IRED. </li><li> 부유 버퍼의 DPS 함유 펠렛을 (50 mM의 HEPES - 코이, 산도 7.5, 150 MM의 염화나트륨, 0.1 mM의 EDTA)를 Resuspend. 2.5 ML로 샘플 볼륨을 조정합니다. </li><li> 버퍼 교환은 PD-10 겔 여과 칼럼을 사용하여 황산 암모늄을 제거합니다. 25 ML의 부유 버퍼에 젤 침대 평형. PD-10 열 상단에 DPS 샘플의 2.5 ML을 적용하고 흐름을 통해 폐기 안으로 흡수 할 수 있습니다. 3.5 ML의 부유 버퍼에 용출 DPS를 수집합니다. </li><li> 4 10 분 16,000 XG에서 버퍼 교환 샘플을 원심 분리기 ° C 모든 불용성 구성 요소를 제거하고, 6-10 ML 희석 버퍼 (50 MM의 HEPES - 코이, 산도 7.5, 0.1 mM의 EDTA)로 희석을 보장하기 위해 그 소금 농도는 DPS가 SP 파로스 열에 바인딩 할 정도로 낮습니다. 초당 특히 농축 시료는 액체의 흐림에 의해 입증 낮은 소금 버퍼에 희석시 작 용해 될 수 있지만, 완전히 작은 amou의 추가에 의해 resolubilized 할 수 있습니다5 M 염화나트륨 용액 NT (추가 10-20 밀리미터). </li><li> SP 버퍼 (50 MM의 HEPES - 코이, 산도 7.5, 50 MM의 염화나트륨, 0.1 mM의 EDTA)와 함께 30 ML SP 세파 빠른 흐름 열 평형. 1.5 ML / 배경 위에 0.3 MPa까지 압력 한계를 가진 분에 열을 버퍼를 실행합니다. 컬럼에 샘플을로드합니다. OD 280 추적이 반환 될 때까지 기준선에 SP 버퍼로 세척하고 흐름을 통해 폐기하십시오. 2 ㎖ 분수를 수집하는 동안, 0 100 % 버퍼 B에서 1​​50 ML 선형 그라데이션을 실행합니다. 10 % 변형이 가능하지만 공격력은 약 50 % B에서 날카로운 피크 용출된다. </li><li> 15 % SDS-PAGE 젤에 용출 분획을 실행하고 분광 광도계를 사용하여 OD 260 / OD 280 비율을 측정하여 DNA의 오염을 확인합니다. 젤은 19 kDa의 주위에 하나의 밴드를 표시해야하며, OD 260 / OD 280 0.7 주위에 일반적이다. 순수한 DPS의 분수 수영장. 와 원심 필터 장치 (예 : Amicon 울트라 여과 장치)를 사용10K 분자량 컷 - 오프 초당 집중하고 저장 버퍼 (50 MM의 HEPES - 코이, 산도 7.5, 50 MM의 염화나트륨)로 교환합니다. -80에서 저장을위한 액체 질소에 분주은 정제 된 DPS 및 동결 ° C. </li></ol> 2. DNA 보호 분석 분석은 여러 시간에 민감한 단계를 포함하고 재현성 결과를 얻기 위해 두 번째로 초과해야한다. 다양한 재료와 피펫 단계가 포함되어 피펫 테이블 (표 1 참조) 단계와 볼륨을 추적하는 것이 좋습니다 때문에. 분석에 필요한 총 시간은 더 이상 3 시간입니다. <ol><li> 밀폐 유리 병에 30 ML 물을 측정, 용액으로부터 산소를 제거하는 10 분 (두 주사기 바늘, 액체의 한, 그 위에 하나를 사용하여)에 대한 N 2와 물을 세척하십시오. 2 밀리미터 주식 솔루션을 얻기 위해 물에 0.0168 g FeSO 4 • 7H 2 O를 추가합니다. 유리 병을 밀봉하고 혼합 한 후, 더 5 분 동안 질소로 세척하십시오. 해결책은해야합니다분명, 노란색의 힌트가 감지되면, 새로운 철 용액을 준비합니다. </li><li> 얼음에 보관하고 알루미늄 호일에 싸서 빛으로부터 보호, H 2 O 2 용액 (1ml를 물에 9.2MH 2 O 2의 10.87 μL)이 100 밀리미터를 확인합니다. 자연 붕괴로 인해 준비 후 1-2 시간 동안 만 사용하십시오. 과산화수소 주식은 부분적으로 적절한 보관 (어둠 속에서, 4 ° C에서)에 의해 개량 할 수있다 고장에 유사하게 적용됩니다. OD 240을 측정하여 주식의 농도를 정기적으로 검사하는 것이 좋습니다. </li><li> 상온의 물을 욕조에 빠르게 초당 정화의 나누어 지는를 해동, 얼음 계속. 침전 집계에 테이블 상단 picocentrifuge 4,000 XG에 10 초 동안 원심 분리기. 분광 광도계 (OD 280 = 1.547 100 μM 단량체 DPS의 농도를 나타낸다)를 사용하여 원심 분리 한 후 농도를 측정한다. </li><li> 12X 반응 버퍼 (1을 사용하여 높은 농도 재고에서 선형 DNA를 희석μL / NG 100의 농도 M MOPS-KOH 산도 7.0, 1 M NaCl을). 선형 DNA를 정량보다 쉽게​​ 사용되지만, 플라스미드 DNA도 함께 사용할 수 있습니다. </li><li> PCR 튜브에 DNA 버퍼 혼합물의 피펫 1 μL. 최종 반응 부피 (마이너스 SDS)은 12 μL 될 수 있도록 반응에 충분한 물을 추가합니다. 이 금액은 피펫 테이블을 사용하여 사전에 계산되어야한다. 3 μM의 최종 dodecamer 농도에 DPS를 추가합니다. DPS는 DNA에 결합 할 수 있도록 실온에서 15 분 알을 품다. </li><li> 원하는 농도에 도달하는 2 개 mm FeSO 4 용액을 추가합니다. 전형적인 실험은 0에서 1 mM의 FeSO 4 농도의 범위를 사용합니다. 높은 농도는 더 광범위한 DNA 저하의 원인이됩니다뿐만 아니라 철은 반응 혼합물에 침전물의 형성으로 이어질 수 있습니다. 빨리 100 개 mm H 2 O 2 용액 (10 mM의 최종 농도) 1 μl를 추가하고, 펜톤 수 있도록, 5 분 실온에서 알을 품다자리를 차지할 매개 분해 반응. </li><li> 20 % SDS (나트륨 도데 실 황산)의 0.8 μl를 추가합니다. 혼합 DPS-DNA 복합체를 방해하는 5 분 동안 85 ° C에서 알을 품다. SDS는 DPS-DNA 복합체를 불안정하게하고 정량의 용이성을 향상 DNA, 원치 않는 젤 변화를 방지 할 수 있습니다. 선택적 단계는 무독성 제품으로 촉매 분해 과산화수소로 반응을 중지하는 것입니다. 이 프로토콜에 설명 된 조건이 단계는 DNA 저하의 넓은 범위를 얻기 위해 필요하지 않습니다. </li><li> 염료를로드 추가 1 분 동안 얼음에 품어와 아가로 오스 겔에로드합니다. 젤을 실행하고 ethidium의 브로마이드를 사용하여 DNA를 얼룩. 젤은 젤 ethidium의 브로마이드 유통을 방해 SDS를 방지하기 위해 사후 전기 스테인드보다는 전기 이전에해야한다. </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+functional+ferritin-like+proteins+with+hydrophobic+cavities.">Design of functional ferritin-like proteins with hydrophobic cavities.</a> Journal of the American Chemical Society. 128 (20), 6611-6619 (2006).</li><li>Ceci, P., Chiancone, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+iron+oxide+nanoparticles+in+Listeria+innocua+Dps+(DNA-binding+protein+from+starved+cells):+a+study+with+the+wild-type+protein+and+a+catalytic+centre+mutant.">Synthesis of iron oxide nanoparticles in Listeria innocua Dps (DNA-binding protein from starved cells): a study with the wild-type protein and a catalytic centre mutant.</a> Chemistry. 16 (2), 709-717 (2010).</li><li>Ceci, P., Ilari, A., Falvo, E., Chiancone, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Dps+protein+of+Agrobacterium+tumefaciens+does+not+bind+to+DNA+but+protects+it+toward+oxidative+cleavage:+x-ray+crystal+structure,+iron+binding,+and+hydroxyl-radical+scavenging+properties.">The Dps protein of Agrobacterium tumefaciens does not bind to DNA but protects it toward oxidative cleavage: x-ray crystal structure, iron binding, and hydroxyl-radical scavenging properties.</a> The Journal of Biological Chemistry. 278 (22), 20319-20326 (2003).</li><li>Su, M., Cavallo, S., Stefanini, S., Chiancone, E., Chasteen, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+so-called+Listeria+innocua+ferritin+is+a+Dps+protein.+Iron+incorporation,+detoxification,+and+DNA+protection+properties.">The so-called Listeria innocua ferritin is a Dps protein. Iron incorporation, detoxification, and DNA protection properties.</a> Biochemistry. 44 (15), 5572-5578 (2005).</li><li>Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+Koelreuteria+paniculata+Laxm.+on+oxidative+stress+and+hydrogen+peroxide-induced+DNA+damage.">Protective effects of Koelreuteria paniculata Laxm. on oxidative stress and hydrogen peroxide-induced DNA damage.</a> Phytopharmacology. 1 (5), 177-189 (2011).</li><li>Stinefelt, B., Leonard, S. S., Blemings, K. P., Shi, X., Klandorf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radical+scavenging,+DNA+protection,+and+inhibition+of+lipid+peroxidation+mediated+by+uric+acid.">Free radical scavenging, DNA protection, and inhibition of lipid peroxidation mediated by uric acid.</a> Annals of Clinical and Laboratory Science. 35 (1), 37-45 (2005).</li><li>Lopez-Gomollon, S., Sevilla, E., Bes, M. T., Peleato, M. L., Fillat, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+role+of+Fur+proteins:+FurB+(All2473)+from+Anabaena+protects+DNA+and+increases+cell+survival+under+oxidative+stress.">New insights into the role of Fur proteins: FurB (All2473) from Anabaena protects DNA and increases cell survival under oxidative stress.</a> The Biochemical Journal. 418 (1), 201-207 (2009).</li><li>Ebrahimi, K. H., Hagedoorn, P. L., Hagen, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+and+stimulation+of+formation+of+the+ferroxidase+center+and+the+iron+core+in+Pyrococcus+furiosus+ferritin.">Inhibition and stimulation of formation of the ferroxidase center and the iron core in Pyrococcus furiosus ferritin.</a> Journal of Biological Inorganic Chemistry. 15 (8), 1243-1253 (2010).</li><li>Smith, F. E., Herbert, J., Gaudin, J., Hennessy, D. J., Reid, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+iron+determination+using+ferene+triazine.">Serum iron determination using ferene triazine.</a> Clinical Biochemistry. 17 (5), 306-310 (1984).</li></ol>

우리는 공개 아무것도 없어.

이 문서에 설명 된대로 공격력의 정화 과정은 매우 강력합니다. 순도는 지속적으로 (&gt; 99 %) 고하고있다, 다른 단백질 보이는 밴드로 SDS-PAGE 젤에 표시되지 않습니다. DPS의 매우 높은 농도로 배양 할 때 부분적으로 DNA의 분해에 의해 입증 그럼에도 불구하고, 정제 된 DPS의 일부 일괄 처리, 핵산의 활동이 나타납니다. 이것은 우리가 정화를 통해 제거 할 수 없었던 낮은 농도에서 높은 활성 DNases의 ?...

호기성 생물은 끊임없이 자신의 DNA뿐만 아니라 다른 중요한 생물학적 거대 분자를 손상시킬 수있는 반응성 산소 종으로 주장해야합니다. 산화 손상의 독성을 중화하는 하나의 강력한 도구 굶주린 세포 (DPS)에서 DNA 결합 단백질이다. 굶주린 E.에서 1992 년 발견 이후 대장균 문화 1, 공격력이 박테리아와 archaebacteria 2의 300 개 이상의 종에서 발견되었습니다. 정지 단계 동안 초당 엄청난 상향 조절은 E의 가장 높은 표현 핵 양체 - 관련 단백질을 만든다 기아 상태 3, 4 이하 대장균. 또한, DPS는 기아, 높은 철 농도, 자외선 노출, 열 충격 및 산화 스트레스 5, 6을 (를) 포함하여 많은 다양한 스트레스 동안 세균의 생존과 DNA의 무결성을 모두 보존하기 위해 표시되었습니다. 12 단량체, w의 안정적인 호모 중합체 복합에 구조적, DPS는 자기 동료의 hich은 구형 빈 껍질로 조립합니다. ~ 4.5 nm의 넓은 내부 구멍은 작은 분자 (7)의 통과를 허용 모공을 통해 용매를 외부에 액세스 할 수 있습니다, 그것은 철 8과 같은 광물 금속을 격리 할 수 있습니다. DPS의 보호 효과가 아닌 특정 DNA 바인딩 1, ferroxidase 활동 및 철 저장 8 등의 여러 가지 생화학 적 활동에서 파생됩니다. DPS의 유익한 생화학 활동의 자세한 연구는 먼저 정화가 필요합니다. DPS는뿐만 아니라 다른 단백질 분리뿐만 아니라, 바인드 된 DNA 7에서해야하므로 초당 정화는 정교한 절차입니다. 우리 최적의 정화 과정은 두 이온 교환 컬럼과 황산 암모늄 침전 단계로 구성된, 많은 일반적인 기술을 사용합니다. 고도로 집중된 공격력이 낮은 소금 조건에서 용액 침전 수있는 여러 버퍼 교환이 필요합니다. 일단 DPS 단백질은 정제 된, 그것은 직접 ferroxidase 활동 8, DNA-결합 양론 9, 철 결합 10의 메커니즘을 측정 분석에 적용 할 수 있습니다. 정제 DPS는 다른 잠재적 인 응용 프로그램이 있습니다. DPS의 안정 중공 구형 구조는 단백질 캐비티 내부 11 소수성 입자를 저장하기위한 발판으로 사용되어 반응 챔버 소설 자성 나노 입자에게 12 합성하는 것 같이. 활성 산소로 인한 손상을 중재하는 초당 공격력의 보호 능력은 명확해야하며 직접 DNA 보호 분석 13, 14을 사용하여 설명 할 수 있습니다. 철 펜튼 화학을 통해 H 2 O 2 분해를 촉진 할 때 체외 과정에서이, 라디칼이 생성됩니다. 이러한 활성 산소를 직접 반응에서 DNA 선물을 손상하고 완전하게 높은 농도를 저하시킬 수 있습니다. 두 가지 주요 DPS 활동은 직접 Fent의 효과를 방해 할 수 두에 매개 급진적 인 생산. DPS는 프로세스에 사용할 수있는 과산화수소를 소모 광물을 촉매 철의 농도를 낮춘다. 또한, DNA에 바인딩 초당 잠재적으로 유리기 손상으로부터 물리적으로 보호하고 덜 반응 표면적이 작은 볼륨으로 그것을 응축 수 있습니다. 이 두 속성의 조합은 잘 보호 DPS 활동을 측정하는 목적에 적합한 DNA 보호 분석을합니다. DNA 보호 분석은 매우 다재다능하고 DPS 특성을 넘어 다양한 애플리케이션에 사용될 수 있습니다. 유리기 손상은 세포의 스트레스의 일반적인 형태이고, 많은 다른 단백질과 화학 물질을 중화하는 데 사용됩니다. 유리기 손상에 대한 마커로 DNA의 무결성을 사용하여 분석의 일반적인 원칙은 거의 모든 라디칼 생산 반응 또는 방해를 에이전트와 함께 사용할 수 있습니다. 다른 사람의 사이에서, 분석은 성공적으로 항산화 특성을 결정하는 데 사용되었습니다K.의 수산기 손상 중재 16 요산의 효과를 특성화하고, 모피 전사 조절 단백질 17의 기능에 새로운 통찰력을 얻기 위해 식품 산업 15에 사용의 paniculata 압축을 풉니 다. 발표 논문에서 분석의 다양한 사용에도 불구하고, 우리는 처음으로 많은 연구자에 대한 불필요 힘든 프로세스에 대한 분석을 설정하게되는 많은 최적화 및 문제 해결 단계가 필수 였을 것으로 나타났습니다. 우리는이 문서에서 제시 프로토콜 항목이 장벽을 제거하는 것을 목표로하고있다.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 </tr>
 <tr>
 <td>BL21(DE3)pLysS competent cells</td>
 <td>Promega</td>
 <td>L1195</td>
 </tr>
 <tr>
 <td>pET-17b DNA</td>
 <td>EMD-Millipore</td>
 <td>69663-3</td>
 </tr>
 <tr>
 <td>LB broth powder</td>
 <td>Sigma-Aldrich</td>
 <td>L3022</td>
 </tr>
 <tr>
 <td>Ampicillin sodium salt</td>
 <td>Sigma-Aldrich</td>
 <td>A0166</td>
 </tr>
 <tr>
 <td>Chloramphenicol</td>
 <td>Sigma-Aldrich</td>
 <td>C0378</td>
 </tr>
 <tr>
 <td>IPTG</td>
 <td>Sigma-Aldrich</td>
 <td>I6758</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>BDH</td>
 <td>441476L</td>
 </tr>
 <tr>
 <td>Potassium hydroxide</td>
 <td>Merck</td>
 <td>105033</td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>VWR</td>
 <td>443824T</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Sigma-Aldrich</td>
 <td>E9884</td>
 </tr>
 <tr>
 <td>Protease Inhibitor Cocktail Set III</td>
 <td>EMD-Millipore</td>
 <td>539134</td>
 </tr>
 <tr>
 <td>DEAE-Sepharose</td>
 <td>Sigma-Aldrich</td>
 <td>DFF100</td>
 </tr>
 <tr>
 <td>Ammonium sulphate</td>
 <td>Sigma-Aldrich</td>
 <td>A4418</td>
 </tr>
 <tr>
 <td>PD-10 Desalting Columns</td>
 <td>GE Healthcare LS</td>
 <td>17-0851-01</td>
 </tr>
 <tr>
 <td>SP-Sepharose</td>
 <td>Sigma-Aldrich</td>
 <td>S1799</td>
 </tr>
 <tr>
 <td>Amicon Ultra Centr. Filter (10K MWCO)</td>
 <td>Millipore</td>
 <td>UFC901024</td>
 </tr>
 <tr>
 <td>Ferrous Sulphate heptahydrate</td>
 <td>Sigma-Aldrich</td>
 <td>F8048</td>
 </tr>
 <tr>
 <td>Hydrogen peroxide solution</td>
 <td>Sigma-Aldrich</td>
 <td>216763</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>Calbiochem</td>
 <td>475898</td>
 </tr>
 <tr>
 <td>SDS solution</td>
 <td>Bio-Rad</td>
 <td>161-0418</td>
 </tr>
 <tr>
 <td>Ethidium bromide</td>
 <td>Sigma-Aldrich</td>
 <td>E1510</td>
 </tr>
 <tr>
 <td>Equipment</td>
 <td>Company</td>
 <td>Model</td>
 </tr>
 <tr>
 <td>Static incubator</td>
 <td>Hettich</td>
 <td>INE500</td>
 </tr>
 <tr>
 <td>Shaking Incubator </td>
 <td>New Brunswick Sc.</td>
 <td>Inova 44</td>
 </tr>
 <tr>
 <td>Cooled centrifuge</td>
 <td>Beckman Coulter</td>
 <td>Avanti J-E</td>
 </tr>
 <tr>
 <td>Table-top centrifuge</td>
 <td>Eppendorf</td>
 <td>5424</td>
 </tr>
 <tr>
 <td>Cell disrupter</td>
 <td>Constant Systems Ltd.</td>
 <td>TS2/40/AA/AA</td>
 </tr>
 <tr>
 <td>FPLC Purifier</td>
 <td>General Electric</td>
 <td>AKTA</td>
 </tr>
 <tr>
 <td>Airtight vials</td>
 <td>Cole-Parmer</td>
 <td>EW-08918-85</td>
 </tr>
 <tr>
 <td>Syringe needles</td>
 <td>BD</td>
 <td>305128</td>
 </tr>
 <tr>
 <td>Pipettes</td>
 <td>Eppendorf</td>
 <td>Z683779-1EA, Z683795-1EA</td>
 </tr>
 </tbody>
</table>

application of an in vitro dna protection assay to visualize stress mediation properties of the dps protein

산화 스트레스는 유산소 생활의 피할 수없는 부산물이다. 산소 분자 지상파 신진 대사를 위해 필수적이며,뿐만 아니라 살아있는 유기체 내의 많은 손상 반응에 참여합니다. 삶에 대한 또 다른 중요한 화합물이다 호기성 대사와 철의 결합은 펜톤 화학을 통해 래디 칼을 생산하고 세포 구성 요소를 분해하기 위해 충분하다. DNA 수리 멀리 사소한에서 그대로 DNA 저하, 틀림없이 세포 내 활성 산소를 포함하는 가장 유해한 프로세스입니다. 이 문서에서 제시된 분석은 급진적 인 매개 DNA 손상에 대한 분자와 효소의 효과를 측정하고 시각화하는 정량 기법을 제공합니다. DNA 보호 분석은 단백질이나 화학 물질의 보호 속성의 체외 특성화, 간단한 빠르고 강력한 도구입니다. 그것은 손상 산화 반응에 DNA를 노출하고 그 화합물의 농도를 다양하게 추가 포함. 화합물의 농도의 함수로 DNA 손상의 감소 또는 증가는 다음 겔 전기 영동을 사용하여 시각화합니다. 이 문서에서 우리는 굶주린 세포 (DPS)에서 DNA-결합 단백질의 보호 특성을 측정하여 DNA 보호 분석의 기법을 보여줍니다. 공격력이 강력하게 환경 스트레스에 대처하기 위해 300 개 이상의 박테리아에 의해 이용되는 미니 페리틴입니다. 여기에서 우리는 DPS 정화 프로토콜과 DPS에 의해 DNA의 보호를 평가하기위한 최적의 분석 조건을 제시한다.

여기에 설명 된 DPS의 정화 과정은 매우 재현. 설명 프로토콜에 따라 DPS 정화, E. 2 L을 사용하여 시작 지점으로 대장균 문화, 일반적으로 5, 12 μM 사이의 농도에서 12 초당을 포함하는 단백질 2.5 mL를 얻을 것입니다. 긴 유도 회 (4 시간)이 변동성을 줄일 것으로 보인다. 단백질 순도는 SDS-PAGE 젤 (그림 1)에 의해 입증, 99 % 이상 일관되게됩니다. DNA 오염의 수준은 모두 DN...

Watch this Scientific Journal Video about Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein at JoVE.com

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

굶주린 세포 (DPS)에서 DNA-결합 단백질은 세균의 스트레스를 퇴치에 중요한 역할을한다. 이 문서의 정화에 대해 설명합니다<em&gt; E. 대장균</em&gt; 공격력과에 대한 프로토콜<em&gt; 체외에서</em&gt; 분석은 활성 산소에 의해 분해로부터 DNA의 DPS - 중재 보호를 시연.

의 적용<em&gt; 체외</emDPS 단백질의 스트레스 중재 속성을 시각화&gt; DNA 보호 분석

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

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Biology

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

11.1K 조회수.  Delft University of Technology. 굶주린 세포 (DPS)에서 DNA-결합 단백질은 세균의 스트레스를 퇴치에 중요한 역할을한다. 이 문서의 정화에 대해 설명합니다 E. 대장균 공격력과에 대한 프로토콜 체외에서 분석은 활성 산소에 의해 분해로부터 DNA의 DPS - 중재 보호를 시연.

비디오: Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

체외에서 FluoroBlok 종양의 침략 분석

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

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생물학

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover1,2.
Here, we report application of stopped-flow kinetics to probe the mechanism of action of Msh2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR)3-5. In doing so, Msh2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from ~10-6 to10-9 bases), and thus helps preserve genomic integrity. Not surprisingly, defective human Msh2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers6-8. In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of Msh2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing Msh2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. DNA dissociation is measured by mixing pre-formed Msh2-Msh6 G:T(2-Ap) mismatch complex with unlabeled trap DNA and monitoring decrease in fluorescence over time9. Pre-steady state ATPase kinetics are measured by the change in fluorescence of 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled Phosphate Binding Protein (MDCC-PBP) on binding phosphate (Pi) released by Msh2-Msh6 following ATP hydrolysis9,10.
The data reveal rapid binding of Msh2-Msh6 to a G:T mismatch and formation of a long-lived Msh2-Msh6 G:T complex, which in turn results in suppression of ATP hydrolysis and stabilization of the protein in an ATP-bound form. The reaction kinetics provide clear support for the hypothesis that ATP-bound Msh2-Msh6 signals DNA repair on binding a mismatched base pair in the double helix.
F. Noah Biro and Jie Zhai contributed to this paper equally.

Msh2-Msh6 is responsible for initiating repair of replication errors in DNA. Here we present a transient kinetics approach towards understanding how this critical protein works. The report illustrates stopped-flow experiments for measuring the coupled DNA binding and ATPase kinetics underlying Msh2-Msh6 mechanism of action in DNA repair.

DNA 복구 단백질의 행동의 메커니즘을 조사하기 위해 중지 흐름 속도론 방법의 응용

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1 On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2 Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3 To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.

We present two independent, microscope-based tools to measure the induced nuclear and cytoskeletal deformations in single, living adherent cells in response to global or localized strain application. These techniques are used to determine nuclear stiffness (i.e., deformability) and to probe intracellular force transmission between the nucleus and the cytoskeleton.

계면 세포 핵의 기계적 성질을 알아내기 위해 Biophysical Assays : 기판 스트레인 신청 및 Microneedle 조작

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

갑각류와 장기 치명적인 독성 시험 Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

체외 메틸화 분석에서 단백질 아르기닌 메틸화를 공부합니다

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

전신 산화 스트레스의 간접 측정으로 혜성 분석

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

만성 후 마우스 비장 세포에서 DNA 손상 및 복구를 측정<em&gt; 생체</em&gt; 베타와 감마 방사선의 매우 낮은 복용량에 노출

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

애기 장대 단백질 DNA 상호 작용의 식별을위한 염색질 면역 침전 분석<em&gt; 생체</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

총회와 &#39;전단 반지&#39;의 응용 프로그램 : 궤도, 단방향 및 정기 유체 흐름 및 전단 응력에 대한 소설 내피 모델

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

파인 우드 선충의 관측 및 짝짓기 행동의 정량화,<em&gt; 소나무 재선충</em

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...