저자는 영장류 식물 성 식품의 에스트로겐 활동을 결정하기 위해 일시적인 과실 작용 작용의 사용에 초기 교육에 대한 데일 라이트먼에 감사드립니다. 브래드포드 웨스트리치와 C. 에릭 존슨 덕분에 실험실 장비를 설치하고 학생들을 추출 방법으로 교육하는 데 도움을 주었습니다. 마지막으로, 이 연구에 자금을 지원해 주신 인디애나 대학교에 감사드립니다.

1. 식물 재료의 준비
<ol><li>리오필라이저를 사용하여 신선한 수거된 건조 식물 아이템을 동결하십시오.<ol>
<li>빛으로부터 샘플을 보호하기 위해 건조 공정 중에 알루미늄 호일로 챔버를 덮습니다.</li>
		<li>샘플이 완전히 건조하도록 하기 위해 챔버가 더 이상 차갑게 느껴지지 않고 계량시 더 이상 질량을 잃지 않도록 합니다.</li>
		<li>분쇄 될 때까지 빛이없는 멸균 낮은 잔류 물 봉투에 말린 식물을 저장합니다.</li>
	</ol>
</li>
	<li>0.85mm 메쉬 스크린을 갖춘 분쇄 공장을 사용하여 샘플을 미세하게 연마합니다.<ol>
<li>추출 될 때까지 빛이없는 가방에 접지 샘플을 저장합니다.</li>
	</ol>
</li>
</ol>2. 식물 이차 대사 산물 추출
<ol><li>이차 식물 대사 산물을 추출하려면 HPLC 급 메탄올의 10 mL에 건조 된 샘플 1 g의 비율을 사용합니다.<ol>
<li>분석 균형에 샘플을 무게와 적절한 크기의 Erlenmeyer 플라스크 (125 – 250 mL)에 추가합니다. 그런 다음 메탄올의 적절한 볼륨을 추가합니다. 추출된 샘플의 질량을 기록합니다.</li>
		<li>알루미늄 호일로 식물 메탄올 용액을 덮은 다음 궤도 셰이커에서 3일 동안 실온(RT)에서 100rpm 속도로 회전하여 잠재적으로 에스트로겐 화합물이 메탄올에 용해될 수 있도록 설정합니다.</li>
		<li>필터 용지(125mm)를 사용하여 상체를 드립 여과 시스템에 넣습니다.</li>
		<li>회전 증발기는 시료가 두꺼워질 때까지 식물 추출물을 건조하지만 300mL 의 둥근 바닥 플라스크에 붓습니다. 샘플을 50mL 원형 플라스크에 붓고, 소량의 메탄올로 큰 플라스크를 헹구세요. 메탄올이 완전히 증발 될 때까지 작은 플라스크에서 샘플을 계속 건조시.</li>
		<li>분석 균형을 사용하여 샘플 잔류물을 계량합니다. 잔류물 질량을 기록합니다.</li>
		<li>DMSO의 2mL에 0.1 g의 추출물 농도로 디메틸 설산화물(DMSO)에 식물 추출물을 용해한다. 균질화 될 때까지 소용돌이.</li>
		<li>식물 추출물-DMSO 용액을 호박유리 바이알에 4°C에 저장하여 분석할 때까지 보관한다.</li>
	</ol>
</li>
</ol>주의: 식물은 알려지지 않은 생물학적 활성 화학 물질을 생산할 수 있으며, DMSO는 세포막을 통해 운반할 수 있는 차량입니다. 이러한 샘플을 취급할 때 적절한 개인 보호 장비와 관리를 사용하십시오.
3. 인간 에스트로겐 수용체 β 트랜스페션 분석12
참고: 무균 기술과 라미나르 플로우 후드는 분석 프로토콜의 1일째에 필요합니다.
<ol><li>표준 곡선을 위해 17β-Estradiol의 희석을 준비합니다.<ol>
<li>37°C 수조에서 냉동고 저장에서 세포 회수 배지 및 화합물 스크리닝 배지(CSM)를 전송합니다.</li>
		<li>라벨 마이크로 센심 분리기 튜브 중간 1 및 2 (INT1, INT2) 및 1-8.</li>
		<li>CSM의 995 μL, CSM의 615 μLINT2, CSM의 900 μL을 가진 튜브 1, 그리고 600 μL의 튜브 2-8로 INT1을 채웁니다. 튜브 8을 따로 설정합니다.</li>
		<li>100 μM 17β-Estradiol 스톡의 5 μL을 INT1로 옮기. 팁을 삭제합니다. 소용돌이.</li>
		<li>각 전송 전에 파이펫을 3번 헹구고 INT1에서 INT2로 10 μL을 전송합니다. 팁을 삭제합니다.</li>
		<li>파이펫을 3번 헹신 다음 INT2에서 100 μL을 튜브 1로 옮킨다. 팁을 폐기합니다. 튜브 1에서 튜브 2로 300 μL을 옮기습니다. 튜브 3에서 7까지 반복하십시오. 튜브 7에서 300 μL을 폐기하여 폐기물 용기에 넣습니다. 튜브 8은 제로이며 에스트라디올을 받지 않습니다. 도금 기준의 최종 농도는 400, 133.3, 44.44, 14.815, 4.938, 1.646, 0.5487 및 0 pM estradiol입니다.</li>
	</ol>
</li>
	<li>샘플 화합물을 준비합니다.<ol>
<li>소용돌이 샘플.</li>
		<li>DMSO의 각 식물 샘플의 4 μL을 취하고 CSM의 496 μL에 추가하여 0.8 % DMSO 솔루션을 생성합니다.</li>
	</ol>
</li>
	<li>기자 셀을 급속히 해동합니다.<ol>
<li>37°C 수조에서 세포 회수 배지의 튜브를 회수합니다. 70% 에탄올을 사용하여 외부 표면을 소독합니다.</li>
		<li>-80°C 저장에서 리포터 셀을 검색하고 냉동 셀의 튜브로 미리 따뜻워진 CRM의 10mL를 전송하여 해동합니다.</li>
		<li>기자 세포의 튜브를 닫고 5-10 분 동안 37 ° C 수조로 옮습니다.</li>
		<li>수조에서 리포터 셀 서스펜션의 튜브를 검색합니다. 세포의 튜브를 여러 번 부드럽게 반전하여 세포의 응집체를 분해하고 균일한 현탁액을 생성합니다. 70% 에탄올로 튜브 표면을 청소합니다.</li>
	</ol>
</li>
	<li>분석 도금<ol>
<li>다중 채널 파이펫을 사용하여 리포터 셀 서스펜션의 100 μL을 각 우물에 분배합니다.</li>
		<li>시료 100μL을 삼중시로 분배하여 적절한 분석 우물에 넣습니다.</li>
		<li>플레이트를 37°C로 옮기고, 22-24h의 가습5%CO2 인큐베이터로 옮김한다.</li>
	</ol>
</li>
	<li>2일째를 준비하기 위해 밤새 어두운 냉장고에서 검출 기판 및 검출 버퍼를 해동합니다.</li>
	<li>플레이트 인큐베이션이 끝나기 직전에 냉장고에서 검출 기판 및 검출 버퍼를 제거하고 RT에 평형될 때까지 저조도 영역에 배치합니다. RT에서 한 번, 철저하게 솔루션을 혼합하기 위해 부드럽게 여러 번 각 튜브를 반전.<ol>
<li>인큐베이션이 완료되기 직전에 검출 버퍼의 전체 내용을 검출 기판 튜브에 부어 루시파아제 검출 시약을 만듭니다. 거품을 생산하지 않도록 부드럽게 섞습니다.</li>
		<li>인큐베이션이 완료되면 플레이트를 뒤집어 콘텐츠를 적절한 폐기물 용기에 버리십시오. 깨끗한 흡수성 종이 타월에 접시를 부드럽게 두드려 우물에서 마지막 물방울을 제거합니다.</li>
		<li>각각의 우물에 루시파아제 검출 시약의 100 μL을 추가합니다. 분석 접시를 RT에서 15분 동안 쉬게 하십시오. 접시를 흔들지 마십시오.</li>
	</ol>
</li>
	<li>96웰 플레이트 판독 광등계를 사용하여 발광을 정량화합니다.</li>
</ol>

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저자는 공개 할 것이 없습니다.

개별적으로 검사하는 의약품 제제에 개발된 ERβ 리포터 분석체는 ERβ를 통해 생물학적으로 활성된 식물성 에스트로겐을 위한 식물성 식품을 선별하는 데도 적합하다. 프로토콜에서 중요한 고려 사항은 식물 샘플을 신중하게 치료하는 것을 포함합니다 : 신선한 식물 재료는 성형 또는 기타 생물학적 저하를 방지하기 위해 신속하게 건조되어야하며 화합물(18)의광분해를 방지하기...

식물은 생존, 번식, 성장, 발달 및 행동1에중요한 칼로리와 영양소를 제공하는 많은 동물에게 필요한 식량 공급원입니다. 식물은 수천 개의 화학 물질을 생산하며, 많은 화학 물질은 자체 성장, 금식 유지 보수 및 재생을 위한 적응으로 많은 것을 생산합니다. 다른 화합물, 간주 식물 이차 대사 산물 (PSM), 덜 명확한 기능을 가지고, 일부는 독성과 가능성이 약초 및 기생에 대한 방어로 사용하지만 (예를 들어, 알칼로이드, 탄닌)2,3. 이러한 화학 물질 중 일부는 내분비 기능과 같은 동물의 장기 생리 적 과정에 영향을 미칠 수있는 능력을 가지고 있지만, 이러한 내분비 활성 식물화학 물질이 척추 동물 내분비 시스템과 상호 작용하는 이유는 여전히 불분명2,4.
가장 잘 연구된 내분비 활성 식물화학 물질인 식물성 내스트로겐은 구조적으로 나기능적으로 에스트로겐을 모방하여 척추동물 내분비 시스템5의hypothalomo-뇌하수체 고나달 축과 직접 상호 작용하는 폴리페놀식 PSM입니다. 인간 규정식에 있는 식물성 에스트로겐의 섭취는 몇몇 암, 심장병 및 갱년기 현상에 대하여 보호와 연관됩니다, 그밖 효력은 불임 문제를 포함하더라도. 사실, 이러한 화합물의 생리적 효과는 1940년대에 양에서 불임이 식물에스트로겐이 풍부한 클로버(Trifolium지하)에방목된 것으로 인해발견되었습니다. 섭취 할 때, 식물성 에스트로겐은 세포로 전달하고 에스트로겐의 효과를 모방 할 수 있습니다. 식물성 에스트로겐은 양 불임에 부정적인 영향을 주었지만, 식물성 에스트로겐과 생리학 사이의 관계는 간단하지 않습니다. 양처럼, 남부 흰 코뿔소는 대두와 알팔파의 다량에서 파생 된 사료에 에스트로겐 화합물에 감도를 표시합니다. 임신 중이 다이어트를 먹이 여성의 딸은7을재현 할 가능성이 적습니다. 그러나, 다른 연구는 식물성 에스트로겐뿐만 아니라 긍정적 인 효과를 가질 수 있음을 보여 주었다, 이전 마우스에서 난소 여포의 성숙을 포함8,특정 암의 예방, 항산화 활성, 및 항증식 효과9.
식물성 에스트로겐의 효과의 폭은 에스트로겐이 생식 및 중추 신경계의 성장, 발달 및 조절을 포함한 다양한 생물학적 기능에 영향을 미친다는 점을 감안할 때 놀라운 일이 아니다10. 행동의 많은 메커니즘이 있지만, 식물에스트로겐은 종종 수정 하는 능력을 가지고, 강화, 또는 핵 내 에스트로겐 수용 체 알파 와 베타에 대 한 리간드로 작동 하는 그들의 능력을 통해 에스트로겐 신호를 방해 (ERα 및 ERβ). 많은 식물성 에스트로겐은 에스트로겐 수용체를 결합 할 수 있도록 에스트로겐과 유사한 페놀 링 구조를 가지고 있습니다. 에스트로겐과 같은 고뇌에 탄성 적 활성 기능을 가진 사람들은 에스트로겐 반응 요소 (ERE)에 이질하고 결합할 수 있는 활성화된 ER-리간드 복합체를 형성하고 유전자 전사를 유발한다11. 따라서, 에스트로겐과 식물성 에스트로겐은 전사 요인으로 그들의 행동을 통해 세포 활동 및 시스템 기능을 조절합니다.
여기서 우리는 에스트로겐성 생물학적 활성을 갖는 화합물의 존재를 위해 식물 추출물을 선별하기 위해 세포 기반 분석의 새로운 사용을 제시합니다. 이 분석은 ERE프로모터(12)에연결된 반딧불(Photinuspyralis)루시파라제 유전자로 전염된 ERβ를 높게 발현하도록 설계된 중국 햄스터 난소 CHO 세포를 사용한다. 에스트로겐 화합물이 존재할 때, 그들은 ER에 결합, 이질, 그리고 ERE에 결합, 루시 파 제 유전자의 전사로 이어지는. 기판 용액을 추가하면, 루시파라제는 광자 방출로 이어지는 반응을 촉매합니다. 따라서 양수 샘플은 빛을 생성하고 음의 샘플은 생성하지 않습니다.
이러한 시판상 분석은 항성 및 용이성에서 불안정하고 가변적이었던 리포터 유전자 및 에스트로겐수용체(13,14)로포유류 세포를 이식할 실험실의 필요성을 제거한다. 분석법은 식물이 수용체 결합을 통해 에스트로겐 활성을 가지고 있는지 여부를 신속하고 간단하게 결정할 수있는 안정적인 트랜스펙트 플랫폼을 제공합니다.
우리는 콩이 현지 식료품에서 인간의 음식을 사용하여 에스트로겐 이소플라본15의 알려진 농도주어진 다른 모든 식품보다 더 높은 에스트로겐 활성을 가지고 가설을 테스트합니다.

<table><tbody><tr><td>1000 &amp;micro;L pipette</td><td></td><td></td><td></td></tr><tr><td>20 &amp;micro;L pipette</td><td></td><td></td><td></td></tr><tr><td>200 &amp;micro;L pipette</td><td></td><td></td><td></td></tr><tr><td>37 &amp;#176; water bath</td><td></td><td></td><td></td></tr><tr><td>37 &amp;#176;, humidified 5% CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td></td><td></td><td></td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>analytical balance</td><td></td><td></td><td></td></tr><tr><td>cell culture-rated laminar flow hood</td><td></td><td></td><td></td></tr><tr><td>dimethyl sulfoxide</td><td></td><td></td><td></td></tr><tr><td>disposable media basin, sterile</td><td></td><td></td><td></td></tr><tr><td>drip filtration system</td><td></td><td></td><td></td></tr><tr><td>Erlenmeyer flasks</td><td></td><td></td><td>125 mL and 250 mL</td></tr><tr><td>HPLC grade methanol</td><td></td><td></td><td></td></tr><tr><td>Human ER&amp;beta; Reporter Assay System, 1 x 96-well format assays</td><td>Indigo Biosciences</td><td>IB00411</td><td>Assay kit - analyzes 24 samples plus standard curve</td></tr><tr><td>lyophilizer</td><td></td><td></td><td></td></tr><tr><td>multi-channel pipette</td><td></td><td></td><td></td></tr><tr><td>orbital shaker</td><td></td><td></td><td></td></tr><tr><td>plate-reading luminometer</td><td></td><td></td><td>ex. Bioteck Synergy HTX</td></tr><tr><td>rotory evaporator</td><td></td><td></td><td></td></tr><tr><td>round bottom flasks</td><td></td><td></td><td>50 mL and 300 mL</td></tr><tr><td>sterile microcentrifuge tubes or sterile multi-channel media basins</td><td></td><td></td><td></td></tr><tr><td>sterile tips</td><td></td><td></td><td>200 &amp;micro;L and 1000 &amp;micro;L</td></tr><tr><td>Whatman grade 1 paper</td><td></td><td></td><td></td></tr><tr><td>whirl-pak bags</td><td></td><td></td><td>sterile polyethylene bags</td></tr></tbody></table>

screening for phytoestrogens using a cell-based estrogen receptor  &#946; reporter assay

식물은 많은 동물을위한 음식의 원천이며 수천 개의 화학 물질을 생산할 수 있습니다. 이러한 화합물 중 일부는 내 분 비 기능 등 그들을 소비 하는 척추 동물에 생리 적 과정에 영향을. 가장 잘 연구된 내분비 활성 식물화학 물질인 식물성 물질은 척추동물 내분비 시스템의 시상라모-뇌하수체 골나달 축과 직접 상호 작용합니다. 여기서 우리는 에스트로겐성 생물학적 활성을 갖는 화합물의 존재를 위해 식물 추출물을 선별하기 위해 세포 기반 분석의 새로운 사용을 제시합니다. 이 분석체는 고도의 발현 에스트로겐 수용체 베타 (ERβ)로 설계된 포유류 세포를 사용하고 루시파아제 유전자로 감염되었습니다. 에스트로겐 활성 화합물에 노출 빛을 생산 하는 세포 결과. 이 분석은 생물학적 에스트로겐 성 활동을 테스트하는 신뢰할 수 있고 간단한 방법입니다. 그것은 일시적인 과도 분석, 특히, 사용의 용이성, 세포의 안정성 및 분석의 감도에 대한 몇 가지 개선이 있습니다.

인간의 식단에서 흔히 발견되는 과일과 채소의 22추출물은 에스트로겐 화합물의 존재를 위해 선별되었습니다. 완두콩 가족은 식물성 식물성 식품16의알려진 공급원뿐만 아니라 무화과, 날짜, 옥수수, 당근, 사과, 바나나, 딸기, 토마토, 케일 및 양배추와 같은 콩류를 포함한 다양한 식품을 분석했습니다. 내분비 방해 화합물은 일반적인 물질 (예를 들어, 플라스틱 및 살충제)에서 ?...

Watch this Scientific Journal Video about Screening for Phytoestrogens using a Cell-based Estrogen Receptor  Î² Reporter Assay at JoVE.com

Screening for Phytoestrogens using a Cell-based Estrogen Receptor  &#946; Reporter Assay

우리는 에스트로겐 활성에 대한 인간 및 비 인간 영장류 식품을 선별하기위한 기자 분석 β 시판 가능한 에스트로겐 수용체를 최적화했습니다. 우리는 알려진 에스트로겐 성 인간 음식 콩 높은 등록 을 보여 줌으로써이 분석 확인, 다른 식품은 활동을 표시 하지 않는 동안.

세포 기반 에스트로겐 수용체를 사용하여 식물에스트로겐에 대한 검사 β 리포터 분석

Plants are a source of food for many animals, and&#160;they can produce thousands of chemicals. Some of these compounds affect physiological processes in ...

screening-for-phytoestrogens-using-cell-based-estrogen-receptor

Research

JoVE Journal

Biochemistry

4.8K 조회수.  Indiana University. 우리는 에스트로겐 활성에 대한 인간 및 비 인간 영장류 식품을 선별하기위한 기자 분석 β 시판 가능한 에스트로겐 수용체를 최적화했습니다. 우리는 알려진 에스트로겐 성 인간 음식 콩 높은 등록 을 보여 줌으로써이 분석 확인, 다른 식품은 활동을 표시 하지 않는 동안.

비디오: Screening for Phytoestrogens using a Cell-based Estrogen Receptor  β Reporter Assay

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

폐수 처리 프로세스의 효능의 평가를위한 화학 및 생체 독성 방법의 배터리를 사용하면 에스트로겐 효능을 제거하는 방법

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

연구

환경과학

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

물 사용에 내분비 활동에 대한 선별 시판<em&gt; 체외</em&gt; 전이 활성화 생물학적 정량

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among species. It has been thought that the function of human and mouse ER&#945;s is identical. We demonstrate the differential 4-hydroxy-tamoxifen (4OHT) effect on mouse and human ER&#945; ligand-binding domain (LBD) dimerization activity using the mammalian two-hybrid (M2H) assay. The M2H assay can demonstrate the efficiency of LBD homodimerization activity in mammalian cells, utilizing the transfection of two protein expression plasmids (GAL4 DNA-binding domain [DBD] fusion LBD and VP16 transactivation domain [VP16AD] fusion LBD) and a GAL4-responsive element (GAL4RE) fused luciferase reporter plasmid. When the GAL4DBD fusion LBD and the VP16AD fusion LBD make a dimer in the cells, this protein complex binds to the GAL4RE and, then, activates a luciferase gene expression through the VP16AD dependent transcription activity. The 4OHT-mediated luciferase activation is higher in the HepG2 cells that were transfected with the mouse ER&#945; LBD fusion protein expression plasmids than in the human ER&#945; LBD fusion protein expression plasmid transfected cells. This result suggests that the efficacy of the 4OHT-dependent mouse ER&#945; LBD homodimerization activity is higher than human ER&#945; LBD. In general, the utilization of the M2H assay is not ideal for the evaluation of nuclear receptor LBD dimerization activity, because agonistic ligands enhance the basal level of the LBD activity and that impedes the detection of LBD dimerization activity. We found that 4OHT does not enhance ER&#945; LBD basal activity. That is a key factor for being able to determine and detect the 4OHT-dependent LBD dimerization activity for successfully using the M2H assay. ER&#945; LBD-based M2H assays may be applied to study the partial agonist activity of selective estrogen receptor modulators (e.g., 4OHT) in various mammalian cell types.

We present a method for analyzing the 4-hydroxy-tamoxifen-dependent estrogen receptor alpha ligand-binding domain dimerization activity using the mammalian two-hybrid assay.

포유류 2 하이브리드 분석 결과 사용 하 여 에스트로겐 수용 체 알파의 Ligand 바인딩 도메인 이합체 화 활동을 감지

Estrogen receptor alpha (ER&#945;) is an estrogenic ligand-dependent transcription regulator. The sequence of ER&#945; protein is highly conserved among ...

암 연구학

An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning display increased atherosclerotic lesions in the aorta compared with female mice with intact ovarian function. However, laboratory models involving estrogen-deficient mice with atherosclerosis-prone status are lacking. This deficit is crucial because clinical estrogen deficiency in menopausal women may aggravate the incidence of pre-existing or ongoing lipid disruption and atherosclerosis. In this study, we establish an in vivo estrogen-deficient mouse model by bilateral ovariectomy via a double dorsal-lateral incision in apolipoprotein E (apoE)-/- mice. We then compare the effects of 17&#946;-estradiol and pseudoprotodioscin (PPD) (a phytoestrogen) perorally administered via hazelnut spread. We find that although PPD exerts some effect on reducing final body weight and plasma TG in OVX apoE-/- mice, it has anti-atherosclerotic and cardiac-protective capacities comparable with its 17&#946;-estradiol counterpart. PPD is a phytoestrogen that has been reported to exert anti-tumor properties. Thus, the proposed method is applicable for screening phytoestrogens via peroral administration to substitute for traditional hormone replacement therapy in postmenopausal women, which has been reported to have potentially deleterious tumorigenetic capacity. Peroral administration via hazelnut spread is noninvasive, rendering it widely applicable to many patients. This article contains step-by-step demonstrations of bilateral ovariectomy via the double dorsal-lateral incision in apoE-/- mice and peroral 17&#946;-estradiol or phytoestrogen hormone replacement via hazelnut spread. Plasma lipid and cardiovascular function analyses using echocardiography follow.

Clinically, estrogen deficiency in menopausal women may aggravate the incidence of lipid disruption and atherosclerosis. We established an in vivo estrogen deficiency model by bilateral ovariectomy via a double dorsal-lateral incision in apoE-/- mice. The mouse model is applicable for screening exogenous estrogen treatments of cardiovascular dysfunction after menopause.

폐경 후 심장 혈관 기능 장애의 외인성 에스트로겐 치료를 선별하기위한 생체 내 에스트로겐 결핍 마우스 모델

Postmenopausal women are at greater risk of developing cardiovascular diseases than premenopausal women. Female mice ovariectomized (OVX) at weaning ...

생화학

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

단일 분자 물고기에 의한 전사 활성 알레일의 단일 세포 분석

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

유방암 세포에서 에스트로겐 조절 마이크로 RNA의 프로파일

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

의학

Using 16&#945;-[18F]-Fluoro-17&#946;-Estradiol PET to Visualize Estrogen Receptor &#945; Expression in Human Breast Cancer Xenografts in Female Ovariectomized Mice

To demonstrate how estrogen receptor alpha (ER&#945;) positive breast cancer xenografts may be visualized in BALB/c nude mice using 16&#945;-[18F]-fluoro-17&#946;-estradiol (18F-FES) positron emission tomography (PET), ovariectomized BALB/c nude mice were injected with ER&#945;-positive breast cancer cells (MCF-7, 3 &#215; 106 cells; shoulder [n = 10] or 4th inguinal mammary fat pad [n = 10]) or ER&#945;-negative breast cancer cells (MDA-MB-231, 1 &#215; 106 cells; mammary fat pad [n = 5]). Mice harboring MCF-7 cells received subcutaneous injections of 20 &#181;g of 17&#946;-estradiol (20 &#181;g/20 &#181;L; corn oil:ethanol, 9:1) in the nape of their necks 2 days prior to cell injection, followed by daily injections five times per week for 5 weeks. Tumor volumes were measured according to the formula: (L*W2)/2 (L; length, W; width). Once tumor volumes reached approximately 100 mm3, 17&#946;-estradiol injections were halted 2 days prior to mice receiving 18F-FES for PET imaging to avoid competitive binding with ER&#945;. Upon 18F-FES administration via the lateral tail vein, PET/MRI was performed for 15 min at 1 h to 1.5 h post-injection. 18F-FES uptake was not observed in ER&#945;-negative, MDA-MB-231 tumor-bearing mice. 18F-FES uptake was most pronounced in mice harboring MCF-7 tumors in the shoulder. In MCF-7 tumors grown in the inguinal mammary fat pad, 18F-FES uptake was less visible, as the intestinal excretion pattern of 18F-FES obscured the radioactivity detectable in these tumors. To use 18F-FES PET as a tool to visualize ER&#945; expression in ER&#945;-positive breast xenografts, we demonstrate that the visibility of 18F-FES uptake is clear in tumors located away from the abdominal region of mice, such as in the shoulder.

Here, we demonstrate a protocol to use 16&#945;-[18F]-fluoro-17&#946;-estradiol (18F-FES) positron emission tomography (PET) as a tool to visualize ER&#945; expression in ER&#945;-positive breast xenografts.

16α-[18F]-Fluoro-17β-Estradiol PET를 사용하여 암컷 난소 절제술 마우스의 인간 유방암 이종이식편에서 에스트로겐 수용체 α 발현 시각화

To demonstrate how estrogen receptor alpha (ER&#945;) positive breast cancer xenografts may be visualized in BALB/c nude mice using ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

유방암에서 에스트로겐 수용체 신호 전달에 의해 대사 규제 시스템 생물학

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade &#34;Environmental Estrogens&#34; (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-&#946;-D-galactopyranoside (ONPG) or chlorophenol red-&#946;-D-galactopyranoside (CPRG)) that are cleaved by &#946;-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting, organizing, and analyzing data; constructing and interpreting graphs; and using common laboratory equipment such as micropipettors and spectrophotometers. Thus, implementing this assay encourages students to engage in inquiry-based learning while exploring emerging issues in environmental science and health.

This article presents an optimized yeast estrogen screen for quantifying ligands in Personal Care Products (PCPs) that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates two colorimetric substrate options, a six-day refrigerated incubation for use in undergraduate courses, and statistical tools for data analysis.

학부 교육 연구소에 대 한 효 모 에스트로겐 화면 최적화를 사용 하는 퍼스널 케어 제품에서 Estrogenic Ligands를 검출

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine ...

생물학

Estrogen Receptor-Reporter Activity Assay to Screen Phytoestrogen Estrogenic Activity

Source: Fender, E. et al., <a href="https://app.jove.com/v/61005">Screening for Phytoestrogens using a Cell-based Estrogen Receptor &#946; Reporter Assay.</a> J. Vis. Exp. (2021).
This video describes a cell-based reporter assay to determine the estrogenic activity of plant-derived compounds. A compound with estrogen-like activity mimics its binding to estrogen receptors and drives downstream reporter enzyme gene transcription. The reporter enzyme generates bioluminescence in a reaction, indicating the estrogenic activity of the compound.

에스트로겐 수용체-리포터 활성 분석법은 식물성 에스트로겐 에스트로겐 활성을 스크리닝합니다.

Source: Fender, E. et al., Screening for Phytoestrogens using a Cell-based Estrogen Receptor &#946; Reporter Assay. J. Vis. Exp. (2021).
This video ...