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 border="0" cellpadding="0" cellspacing="0" style="width:721px;" width="722">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">1000 &#181;L pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:308px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">20 &#181;L pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">200 &#181;L pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">37 &#8451; water bath</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:215px;">37 &#8451;, humidified 5% CO2 incubator</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">70% ethanol</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">analytical balance</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">cell culture-rated laminar flow hood</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">dimethyl sulfoxide</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">disposable media basin, sterile</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">drip filtration system</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Erlenmeyer flasks</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>125 mL and 250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">HPLC grade methanol</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Human ER&#946; Reporter Assay System, 1 x 96-well format assays</td>
			<td style="width:80px;">Indigo Biosciences</td>
			<td style="width:119px;">IB00411</td>
			<td>Assay kit - analyzes 24 samples plus standard curve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">lyophilizer</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">multi-channel pipette</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">orbital shaker</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">plate-reading luminometer</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>ex. Bioteck Synergy HTX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">rotory evaporator</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">round bottom flasks</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>50 mL and 300 mL</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">sterile microcentrifuge tubes or sterile multi-channel media basins</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">sterile tips</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td>200 &#181;L and 1000 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Whatman grade 1 paper</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">whirl-pak bags</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></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  ÃŽÂ² 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

4.7K 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

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

소설 불용성 발색 기질 분석 키트를 사용하여 탄수화물 분해 효소의 높은 처리량 검열

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

연구

생화학

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling cascades during homeostasis and disease, and as such represent prime therapeutic targets. Despite their relevance, these interaction networks remain significantly underrepresented in current databases; therefore, most extracellular proteins have no documented binding partner. This discrepancy is primarily due to the challenges associated with the study of the extracellular proteins, including expression of functional proteins, and the weak, low affinity, protein interactions often established between cell surface receptors. The purpose of this method is to describe the printing of a library of extracellular proteins in a microarray format for screening of protein-protein interactions. To enable detection of weak interactions, a method based on multimerization of the query protein under study is described. Coupled to this microbead-based multimerization approach for increased multivalency, the protein microarray allows robust detection of transient protein-protein interactions in high throughput. This method offers a rapid and low sample consuming-approach for identification of new interactions applicable to any extracellular protein. Protein microarray printing and screening protocol are described. This technology will be useful for investigators seeking a robust method for discovery of protein interactions in the extracellular space.

Here, we present a protocol to screen extracellular protein microarrays for identification of novel receptor-ligand interactions in high throughput. We also describe a method to enhance detection of transient protein-protein interactions by using protein-microbead complexes.

세포 외 단백질 Microarray 기술은 낮은 친 화력 수용 체 Ligand 상호 작용의 높은 처리량 검색

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

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 ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

핵산 프로브를 이용한 뉴클레아제 활성의 운동 스크리닝

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

소분자 억제제 스크리닝을 위한 NADH 결합 ATPase 분석의 반 높은 처리량 적응

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn&#39;s disease. A detailed protocol for the induction of Crohn&#39;s disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.

The protocol presents a complete validated TNBS-induced murine model of Crohn&#39;s disease and methods for visualization of estrogen receptors by immunohistochemistry using immunofluorescence of formalin-fixed colon sections embedded in paraffin.

면역 형광을 사용하여 TNBS 유도 크론 병을 가진 마우스의 결장에서 에스트로겐 수용체의 가시화

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

PRESTO-탱고 분석기를 사용하여 GPCR 전체 규모의 리간드 스크리닝을 위한 β-Arrestin2 모집의 병렬 심문

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

접합 효율 모니터링을 위한 기자 기반 셀룰러 분석

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

재조합 G 단백질 결합 수용체의 고처리량 스크리닝을 위한 "이중 첨가" 칼슘 형광 분석

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

<meta charset="utf8"></head><body>비타민 A 수용체 동역학 및 레티노이드 항상성 작용 연구

Methodology for Studying Interactions of Vitamin A Membrane Receptors and Opsin Protein with their Ligands in Generating the Retinylidene Protein

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and generating the retinylidene protein for visual function. RBP4-ROL is the complex of ROL with retinol-binding protein 4 (RBP4), which is present in the blood. Two membrane receptors, Retinol Binding Protein 4 Receptor 2 (RBPR2) in the liver and STimulated by Retinoic Acid 6 Retinol (STRA6) in the eye, bind circulatory RBP4 and this mechanism is critical for internalizing ROL into cells. Establishing methods to investigate receptor-ligand kinetics is essential in understanding the physiological function of vitamin A receptors for retinoid homeostasis. Using Surface Plasmon Resonance (SPR) assays, we can analyze the binding affinities and kinetic parameters of vitamin A membrane receptors with its physiological ligand RBP4. 
These methodologies can reveal new structural and biochemical information of RBP4-binding motifs in RBPR2 and STRA6, which are critical for understanding pathological states of vitamin A deficiency. In the eye, internalized ROL is metabolized to 11-cis retinal, the visual chromophore that binds to opsin in photoreceptors to form the retinylidene protein, rhodopsin. The absorbance of light causes the cis-to-trans isomerization of 11-cis retinal, inducing conformational changes in rhodopsin and the subsequent activation of the phototransduction cascade. Decreased concentrations of serum and ocular ROL can impact retinylidene protein formation, which in turn can cause rhodopsin mislocalization, apoprotein opsin accumulation, night blindness, and photoreceptor outer segment degeneration, leading to Retinitis Pigmentosa or Leber Congenital Amaurosis. 
Therefore, spectrophotometric methodologies to quantify the G protein-coupled receptor opsin-11-cis retinal complex in the retina are critical for understanding mechanisms of retinal cell degeneration in the above-mentioned pathological states. With these comprehensive methodologies, investigators will be able to better assess dietary vitamin A supply in maintaining systemic and ocular retinoid homeostasis, which is critical for generating and maintaining retinylidene protein concentrations in photoreceptors, which is critical for sustaining visual function in humans.

<meta charset="utf8"></head><body>저자 스포트라이트: 비타민 A 수송 메커니즘 규명 - RBPR2 및 RBP4 상호 작용을 통해 간 수용체를 시력 건강에 연결

Here, we describe two quantitative methods for studying the protein-ligand interactions of vitamin A membrane receptors and photoreceptor opsin with their respective physiological ligands.

레티닐리덴 단백질 생성에서 비타민 A 막 수용체 및 옵신 단백질과 리간드의 상호 작용을 연구하기 위한 방법론

Distribution of dietary vitamin A/all-trans retinol (ROL) throughout the body is critical for maintaining retinoid function in peripheral tissues and ...