This work received support through grant HA 6136/1-1 from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) to MH. JAT is supported by Program and Project Grants from the National Health and Medical Research Council of Australia.

وأجريت الطحال كانت مستمدة من الفئران (6-10 أسابيع من العمر) وجميع التجارب على الحيوانات وفقا للمبادئ التوجيهية الأخلاقية الحيوان مركز السرطان ماك كالام بيتر (E486): ملاحظة. 1. إعداد العينات. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد تنشيط خلايا فأر NK. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عزل خلايا NK ساذجة الأولية من وحيدة الخلية تعليق من الطحال من C57BL / 6 أو B6.GrzmB - / - (GzmB اغية الجينات) الفئران عن طريق الانتقاء السلبية باستخدام أدوات متوفرة تجاريا. </li><li style=";text-align:right;direction:rtl"> اتبع بروتوكول الصانعين. لفترة وجيزة، مغناطيسيا تسمية &quot;غير NK-&quot; خلايا مثل T و B الخلايا، والخلايا الجذعية، المحببة، الضامة، وخلايا الدم الحمراء باستخدام الأجسام المضادة البيوتين مترافق ضد علامات سطح الخاصة. استخدام الفصل المغناطيسي مع حبات مكافحة البيوتين في استنزاف خلايا &quot;غير NK&quot;. </li><li style=";text-align:right;direction:rtl"> ثقافة معزولة خلايا NK لمدة 5-7 أيام في وسائل الإعلام التي تحتوي على IL-2 (1،000 U / مل) في 700،000 خلية / مل في 24 لوحات جيدةفي 37 &#160; ° C. </li><li style=";text-align:right;direction:rtl"> بدلا من ذلك، تنشيط خلايا T استخدام 19، وتقييد مستضد الأساسي CTL من OT1 T مستقبلات الخلايا الفئران المعدلة وراثيا 20، ولدت CTL في الثقافات المختلطة اللمفاويات 21، أو supernatants من خلايا NK تنشيط لتحديد نشاط يفرز GzmB 19. </li></ol></li><li style=";text-align:right;direction:rtl"> صيانة الإنسان NK خط الخلية وعزل الخلايا NK الأولية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الحفاظ على سرطان الدم NK البشري (خلايا YT) في معهد روزويل بارك التذكاري (RPMI) متوسطة تستكمل مع 10٪ مصل العجل الجنين. بدلا من ذلك، تنقية الخلايا القاتلة الطبيعية الأولية من الدم المحيطي من المواضيع الصحية وتحفيز لمدة 2-3 أيام في المتوسط ​​تحتوي على IL-2 في 100 U / مل كما هو موضح سابقا (22). </li></ol></li><li style=";text-align:right;direction:rtl"> جيل من الخلية لست]. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> غسل الخلايا ثلاث مرات في برنامج تلفزيوني (الرقم الهيدروجيني 7.4)، ثم وقف في Nonidet P-40 تحلل العازلة (0.1٪ NP-40، 250 مم كلوريد الصوديوم، و 25 ملي HEPES، 2.5 ملي EDTA). من الناحية المثالية، ليز ما لا يقل عن 5 × 10 6 </sتصل&gt; خلايا NK، أو 1 × 10 7 T الخلايا في 100 ميكرولتر عازلة. لا تقم بإضافة مثبطات الأنزيم البروتيني، وكثير وكذلك مثبطات البروتياز سيرين لا رجعة فيها، وعلى وجه الخصوص في tryptases مثل GzmA. احتضان على الجليد لمدة 20 دقيقة، ثم تكوير نوى في microcentrifuge في 15،000 x ج لمدة 10 دقيقة. تجاهل بيليه النووي ونقل طاف لأنبوب جديد. </li></ol></li><li style=";text-align:right;direction:rtl"> تحديد تركيز البروتين من لست]. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام بروتوكول المتاحة تجاريا من اختيار مثل برادفورد أو حمض bicinchoninic (BCA) وتحديد تركيز البروتين. تأكد من أن التركيز هو ~ 2 ملغ كحد أدنى / مل. متجر لست] في -20 درجة مئوية حتى المطلوبة (النشاط granzyme مستقرة لعدة شهور في -20 ° C). </li></ol></li></ol> 2. باء granzyme آخر الفحص <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد الكواشف <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد 10 ملي الأسهم من ركائز (بوك-AAD-S-BZL أو التيار المتردد-IEPD-السلطة الوطنية الفلسطينية) في DMSO (~ 5 ملغ / مل) وتخزينها في aliquots في -20 &amp; #176، C. إعداد تخفيف العمل الطازجة وتجاهل بعد كل يوم، كما بوك-AAD-S-BZL hydrolyzes جزئيا على التخزين في DMSO. </li><li style=";text-align:right;direction:rtl"> إعداد 250 ملي الأسهم من كاشف اللونية (5،5&#39;-ثنائي ثيو؛ ديثيو مكرر (حمض 2-nitrobenzoic)، DTNB) في DMSO (0.09 جم / مل) وتخزينها في -20 ° C. إعداد التخفيف عمل جديدة كل يوم. مطلوب فقط هذا كاشف عن ركائز مع جماعات مؤشر SBzl، وليس السلطة الوطنية الفلسطينية. </li><li style=";text-align:right;direction:rtl"> تشكل عازلة جديدة (0.1M HEPES، 0.05 M MgCl 2، ودرجة الحموضة 7.3) كل 2-3 أسابيع. </li></ol></li><li style=";text-align:right;direction:rtl"> جلب DTNB وركائز الاصطناعية إلى درجة حرارة الغرفة. تمييع ركائز (1:16، على سبيل المثال 100 ميكرولتر في 1.6 مل من العازلة) وDTNB (1: 250، على سبيل المثال 10 ميكرولتر في 2.5 مل عازلة) وتخلط جيدا. </li><li style=";text-align:right;direction:rtl"> تمييع ما لا يقل عن 100 ميكروغرام البروتين المحللة من كل عينة في المنطقة العازلة على الحجم النهائي من 160 ميكرولتر. باستخدام ماصة الأقنية إضافة 50 ميكرولتر لكل بئر في ثلاث نسخ (~ 33.3 ميكروغرام / جيد) في &#39;&#39; U &#39;&#39; الفينيل أسفل لوحة 96-جيدا.تشمل &quot;العازلة فقط&quot; التحكم (أيضا في ثلاث نسخ). </li><li style=";text-align:right;direction:rtl"> إضافة 100 ميكرولتر من المخفف DTNB لسيطرة عينات / عازلة (حذفت هذه الخطوة عند العمل مع ميلان-IEPD-السلطة الوطنية الفلسطينية. ملاحظة: الشق من التيار المتردد-IEPD-السلطة الوطنية الفلسطينية من قبل GzmB النشرات مباشرة الفلورة السلطة الوطنية الفلسطينية (الشكل 4B). </li><li style=";text-align:right;direction:rtl"> استخدام ماصة الأقنية لإضافة بسرعة 50 ميكرولتر من ركائز المخفف إلى كل مجموعة من يثلث، بدءا من المخزن المؤقت ثم أي ضوابط السلبية والتشطيب مع العينات الايجابية المتوقعة. </li><li style=";text-align:right;direction:rtl"> وضع لوحة في قارئ لوحة. قياس النشاط آسيا والمحيط الهادئ-بورصة عمان من قبل التحلل من بوك علاء علاء-ASP-S-BZL / AC-IEPD-السلطة الوطنية الفلسطينية في القارئ صفيحة ميكروسكوبية OD 405 نانومتر. استخدام بروتوكول الفحص الحركي، ويستغرق 25 القراءات، كل 15 ثانية (حوالي 6 دقائق الوقت في القراءة الإجمالية). </li><li style=";text-align:right;direction:rtl"> استخدام قراءات الامتصاصية ولدت في كل نقطة زمنية لحساب معدل الركيزة الانقسام يدويا إذا كان برنامج لوحة القارئ لا يمكن أن تولد قيمة للVMAX. </li></ol> 3. تحليل داتا ملاحظة: البيانات التي تم إنشاؤها يرد كحد أقصى سرعة، الذي يعرف بأنه تغيير OD على مر الزمن (وزارة الدفاع / دقيقة). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لتحليل البيانات، وتحديد الحد الأقصى لمعدل الانقسام الركيزة، والذي يحسب من معدل التغير في الامتصاصية. القيام بذلك تلقائيا باستخدام برنامج قارئ لوحة، التي لديها عادة هذا الخيار. </li><li style=";text-align:right;direction:rtl"> بدلا من ذلك بعد عرض المؤامرات الحركية للتغيير في الامتصاصية، والتي يتم إنشاؤها أثناء الفحص الحركي على قارئ لوحة، يدويا تحديد نقاط البيانات التي تعطي أشد خط على التوالي. بالنسبة لمعظم المقايسات ويتحقق الحد الأقصى لمعدل داخل دقيقة 2 الأول من الفحص. </li><li style=";text-align:right;direction:rtl"> طرح القراءة OD الأولية من أعلى قراءة OD على هذا الخط المستقيم والقسمة على الفاصل الزمني. نقل البيانات الخام إلى منصة مايكروسوفت اكسل / الرسم البياني للتحليل الإحصائي وعرض الرسوم البيانية. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+cytotoxic+T+cell+granule+serine+protease+granzyme+H+has+chymotrypsin-like+(chymase)+activity+and+is+taken+up+into+cytoplasmic+vesicles+reminiscent+of+granzyme+B-containing+endosomes.">The human cytotoxic T cell granule serine protease granzyme H has chymotrypsin-like (chymase) activity and is taken up into cytoplasmic vesicles reminiscent of granzyme B-containing endosomes.</a> J Biol Chem. 274 (43), 30468-30473 (1999).</li><li>Sutton, V. R., 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=Initiation+of+apoptosis+by+granzyme+B+requires+direct+cleavage+of+bid,+but+not+direct+granzyme+B-mediated+caspase+activation.">Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation.</a> J Exp Med. 192 (10), 1403-1414 (2000).</li></ol>

The authors have nothing to disclose.

تاريخيا تم تحديد granzymes كما الجزيئات المستجيب الرئيسية من الخلايا الليمفاوية السامة للخلايا (خلايا CTL وNK) قادرة على إحداث الموت أفكارك السريع في الخلايا المستهدفة. جاء هذا الارتفاع نتيجة لعمل GzmB، التي المشقوق المستهدفة جزيئات الركيزة في اسبارتاتي (D) المخلفات وبالتال?...

Granzymes هي عائلة من البروتياز سيرين وجدت في الجسيمات الحالة إفرازية من القاتلة الطبيعية (NK) الخلايا والخلايا الليمفاوية التائية السامة (CTL) 1. وتوجد خمسة granzymes مختلفة في البشر (A، B، H، K وM)، وعشرة في الفئران (A - G، K، M و N) 2،3. Granzyme ألف وباء granzyme (GzmA، GzmB) هي الأكثر وفرة وتم التحقيق فيها على نطاق واسع في الإعداد البشري والقوارض. وظيفة كلاسيكية من GzmB هو استحثاث موت الخلايا المبرمج في الخلايا المستهدفة تنفيذها بالتزامن مع البروتين perforin تشكيل المسام، مما يسمح للgranzyme للوصول إلى الخلية المستهدفة العصارة الخلوية (4). وعلى الرغم من GzmB التعبير وجدت بشكل لا لبس فيه في الخلايا الليمفاوية السامة للخلايا، والدراسات الأخيرة تم معالجة مجموعة متنوعة من أنواع أخرى خلية، معربا عن GzmB، بما في ذلك ولكن لا تقتصر على الخلايا الكيراتينية 5، قعدات 6، 7 الخلايا البدينة، الخلايا الجذعية بلازماوية الشكل 8، والخلايا B 9، 10.وفي هذا السياق، تم الكشف عن وظائف GzmB غير أفكارك تتراوح من المشاركة في عمليات التهابات، وإعادة عرض الأنسجة وخصائص immunoregulatory أخرى 11-14. وبالنظر إلى أن تم اقتراح دورا البيولوجي أوسع للGzmB مما اعتقد سابقا، تتطلب الباحثين أدوات محددة وموثوق بها للكشف لها. من ميزة هو شرط GzmB المحددة ليلتصق على الجانب الكربوكسيل من بقايا حمض الأسبارتيك، وهي خاصية فريدة من نوعها بين البروتياز سيرين حقيقية النواة 15. الماوس والبشرية وGzmB الفئران وهيكليا مشابهة جدا، ولكن خصوصية الركيزة طويلة من الماوس GzmB تختلف بمهارة من أن كل من الإنسان والفئران 16، وهو ما يعني أن بعض ركائز العامة مع آسيا والمحيط الهادئ في محطة (P1) يمكن المشقوق كفاءة من خلال GzmB من جميع الأنواع الثلاثة، في حين ركائز أخرى مع تسلسل أكثر تعقيدا من المنبع P1 قد تعطي نتائج متفاوتة على نطاق واسع. في كل من الماضي وأكثر من ذلك لى مؤخراterature، هذه الحقيقة قد تسبب ارتباك كبير وسوء فهم لأهمية بيولوجية لبعض النتائج التجريبية، على الرغم تسيطر عليها بعناية، سعت الدراسات الحركية لتصحيح الوضع 17. في هذه الورقة سعينا لتوضيح هذه النقاط باستخدام اثنين من ركائز المتاحة تجاريا، وهي بوك علاء علاء-ASP-SBzl وN-أسيتيل-إيل-حمض الغلوتاميك-برو-ASP-P-nitroanilide. الكواشف اثنين لا تولد مختلف الجماعات المتفاعلة التالية الانقسام (أ sulphydryl مجانا مقابل paranitroanilide مجانا الفلورسنت)، ولكن هذا أي تأثير مهما على انشقاق بروتين. بروتوكول صفها هو التكيف الحديثة من بروتوكول قديمة جدا 18، ولكن ينبغي أن تساعد المحققين لاستخدام ركائز GzmB المختلفة بشكل مناسب، مع توفير إطار منهجي للكشف عن نشاط granzymes أخرى، مثل GzmA وGzmH.

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			<td height="21" style="height:21px;width:284px;">Product</td>
			<td style="width:241px;">Company</td>
			<td style="width:181px;">Catalogue number</td>
			<td style="width:427px;">Comment/description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Boc-Ala-Ala-Asp-S-Bzl</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SMSB05</td>
			<td>Granzyme B substrate (mouse and human)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ac-IEPD-pNA&#160;</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SMPNA009</td>
			<td>Granzyme B substrate (only human)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-a-CBZ-L-lysine-S-Bzl</td>
			<td>Sigma-Aldrich</td>
			<td>C3647</td>
			<td>Granzyme A substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suc-Phe-Leu-Phe-S-Bzl</td>
			<td>SM Biochemicals LLC, CA</td>
			<td>SB025</td>
			<td>Granzyme H substrate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5,5&#8217;-dithio-bis(2-nitrobenzoic acid)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D8130</td>
			<td>&#160;DTNB, Ellman&#8217;s Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NK cell isolation kit II mouse</td>
			<td>Miltenyi Biotec GmbH</td>
			<td style="width:181px;">130-096-892</td>
			<td style="width:427px;">negative selection kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NK cell isolation kit human</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-092-657</td>
			<td style="width:427px;">negative selection kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="21" style="height:21px;">Plate reader</td>
			<td>Biorad iMark</td>
			<td></td>
			<td>Biorad Microplate Manager Software Version MPM6.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serocluster U-bottom vinyl 96-well plate</td>
			<td>Corning, MA, USA</td>
			<td>2797</td>
			<td></td>
		</tr>
	</tbody>
</table>

a colorimetric assay that specifically measures granzyme b proteolytic activity: hydrolysis of boc-ala-ala-asp-s-bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

الركيزة بوك-AAD-S-BZL هي محددة لGzmB وGzmB سيرين البروتياز هو أحد المكونات الرئيسية من الخلايا الليمفاوية السامة للخلايا (خلايا CTL وNK) وغالبية سكانها من المسؤول عن إحداث الموت أفكارك السريع في الخلايا المستهدفة، مثل الخلايا أو ?...

Watch this Scientific Journal Video about A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl at JoVE.com

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

واللونية الفحص أن يقيس على وجه التحديد آخر باء granzyme متعلق ب التحلل البروتيني: التحلل من بوك علاء علاء-ASP-S-BZL

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

a-colorimetric-assay-that-specifically-measures-granzyme-b

Research

JoVE Journal

Chemistry

12.9K Views.  Peter MacCallum Cancer Centre. We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

Video: A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios &lt; 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios &gt; 1.53.
However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at &asymp;280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 &lt; ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

فحوصات اللونية السريعة للتمييز نوعيا RNA والحمض النووي في العينات البيولوجية

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

ثابت للدولة، قبل المطرد للدولة، واحدة دوران قياس الحركية للنشاط الحمض النووي Glycosylase

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

رصد القائم على مضان آخر PAD4 عبر برو-مضان الركيزة النظير

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

Dynamic Electrochemical Measurement of Chloride Ions

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver chloride electrode is used extensively for potentiometric measurement of chloride ions concentration in electrolyte. In this measurement, long-term and continuous monitoring is limited due to the inherent drift and the requirement of a stable reference electrode. We utilized the chronopotentiometric approach to minimize drift and avoid the use of a conventional reference electrode. A galvanostatic pulse is applied to an Ag/AgCl electrode which initiates a faradic reaction depleting the Cl&#713; ions near the electrode surface. The transition time, which is the time to completely deplete the ions near the electrode surface, is a function of the ion concentration, given by the Nernst equation. The square root of the transition time is in linear relation to the chloride ion concentration. Drift of the response over two weeks is negligible (59 &#181;M/day) when measuring 1 mM [Cl&#713;]using a current pulse of 10 Am-2. This is a dynamic measurement where the moment of transition time determines the response and thus is independent of the absolute potential. Any metal wire can be used as a pseudo-reference electrode, making this approach feasible for long-term measurement inside concrete structures.

Dynamic measurement of chloride ions is presented. Transition time of an Ag/AgCl electrode, during a chronopotentiometric technique, can give the concentration of chloride ions in electrolyte. This method does not require a stable conventional reference electrode.

ديناميكية الكهروكيميائية قياس كلوريد الأيونات

This protocol describes the dynamic measurement of chloride ions using the transition time of a silver silver chloride (Ag/AgCl) electrode. Silver silver ...

A Method to Manipulate Surface Tension of a Liquid Metal via Surface Oxidation and Reduction

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where interfacial tension is a dominant force. A variety of methods exist for controlling the interfacial tension of aqueous and organic liquids on this scale; however, these techniques have limited utility for liquid metals due to their large interfacial tension.
Liquid metals can form soft, stretchable, and shape-reconfigurable components in electronic and electromagnetic devices. Although it is possible to manipulate these fluids via mechanical methods (e.g., pumping), electrical methods are easier to miniaturize, control, and implement. However, most electrical techniques have their own constraints: electrowetting-on-dielectric requires large (kV) potentials for modest actuation, electrocapillarity can affect relatively small changes in the interfacial tension, and continuous electrowetting is limited to plugs of the liquid metal in capillaries.
Here, we present a method for actuating gallium and gallium-based liquid metal alloys via an electrochemical surface reaction. Controlling the electrochemical potential on the surface of the liquid metal in electrolyte rapidly and reversibly changes the interfacial tension by over two orders of magnitude ( &#820;500 mN/m to near zero). Furthermore, this method requires only a very modest potential (&#60; 1 V) applied relative to a counter electrode. The resulting change in tension is due primarily to the electrochemical deposition of a surface oxide layer, which acts as a surfactant; removal of the oxide increases the interfacial tension, and vice versa. This technique can be applied in a wide variety of electrolytes and is independent of the substrate on which it rests.

We present a method to control the interfacial energy of a liquid metal in an electrolyte via electrochemical deposition (or removal) of a surface oxide layer. This simple method can control the capillary behavior of gallium-based liquid metals by tuning the interfacial energy rapidly, significantly, and reversibly using modest voltages.

A طريقة لمعالجة التوتر السطحي من المعدن السائل عن طريق الأكسدة السطحية والحد من

Controlling interfacial tension is an effective method for manipulating the shape, position, and flow of fluids at sub-millimeter length scales, where ...

Preparation and Reactivity of a Triphosphenium Bromide Salt: A Convenient and Stable Source of Phosphorus(I)

We present herein the optimized synthesis of a triphosphenium bromide salt. Apart from being a versatile metathesis reagent, this unusually stable low-valent-phosphorus-containing compound acts as a useful P+ transfer agent. Unlike traditional methods employed to access low-coordinate phosphorus species which usually require pyrophoric phosphorus-containing precursors (white phosphorus, Tris(trimethylsilyl)phosphine, etc.), or harsh reducing agents (alkali metals, potassium graphite, etc.), the current approach does not involve pyrophoric or explosive reagents and can be done on large scales (&#62;20 g) in excellent yields by undergraduates with basic air-free synthetic training. The bromide counter ion is readily exchanged with other anions such as tetraphenyl borate (described herein) using typical salt metathesis reagents to obtain materials with desired properties and reactivities. The versatility of this P+ transfer approach is exemplified by the reactions of these triphosphenium precursors with an N-heterocyclic carbene and an anionic bisphosphine, each of which readily displace the neutral bisphosphine to give an NHC-stabilized phosphorus(I) cation and a phosphorus(I) containing zwitterion, respectively.

Preparation and Reactivity of a Triphosphenium Bromide Salt: A Convenient and Stable Source of PhosphorusI

The synthesis of a triphosphenium bromide salt is described and its use as a P+ transfer agent is outlined by reactions with an N-heterocyclic carbene and an anionic bisphosphine, yielding an NHC-stabilized P(I) cation and a P(I) containing zwitterion, respectively.

إعداد والتفاعلية من Triphosphenium بروميد الملح: والمصدر مريحة ومستقرة من الفوسفور (I)

We present herein the optimized synthesis of a triphosphenium bromide salt. Apart from being a versatile metathesis reagent, this unusually stable ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

قياس واحد من HDAC1 والنشاط HDAC6 في الحلة الخلايا باستخدام أوهبلك--مرض التصلب العصبي المتعدد

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation usually consists in the formation of droplets containing low molar mass liquid crystals at elevated temperatures. Subsequently, these particle precursors are oriented in the flow field of the capillary and solidified by a crosslinking polymerization, which produces the final actuating particles. The optimization of the process is necessary to obtain the actuating particles and the proper variation of the process parameters (temperature and flow rate) and allows variations of size and shape (from oblate to strongly prolate morphologies) as well as the magnitude of actuation. In addition, it is possible to vary the type of actuation from elongation to contraction depending on the director profile induced to the droplets during the flow in the capillary, which again depends on the microfluidic process and its parameters. Furthermore, particles of more complex shapes, like core-shell structures or Janus particles, can be prepared by adjusting the setup. By the variation of the chemical structure and the mode of crosslinking (solidification) of the liquid crystalline elastomer, it is also possible to prepare actuating particles triggered by heat or UV-vis irradiation.

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

إعداد موائع جزيئية للمحركات الاستومر البلورية السائلة

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation ...

Synthesis of Esters Via a Greener Steglich Esterification in Acetonitrile

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the reaction is commonly performed using chlorinated or amide solvent systems, which are hazardous to human health and the environment. Our methodology utilizes acetonitrile as a greener and less hazardous solvent system. This protocol exhibits rates and yields that are comparable to traditional solvent systems and employs an extraction and wash sequence that eliminates the need for the purification of the ester product via column chromatography. This general method can be used to couple a variety of carboxylic acids with 1&#176; and 2&#176; aliphatic alcohols, benzylic and allylic alcohols, and phenols to obtain pure esters in high yields. The goal of the protocol detailed here is to provide a greener alternative to a common esterification reaction, which could serve useful for ester synthesis in both academic and industrial applications.

A modified Steglich esterification reaction was used to synthesize a small library of ester derivatives with primary and secondary alcohols. The methodology uses a non-halogenated and greener solvent, acetonitrile, and enables product isolation in high yields without the need for chromatographic purification.

توليف استرات عبر أكثر اخضرارا الأسترة ستيجليتش في الاسيتو الانيتريل

The Steglich esterification is a widely-used reaction for the synthesis of esters from carboxylic acids and alcohols. While efficient and mild, the ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

NMR القائمة على تحليل النشاط لتحديد تثبيط مركب, قيم IC50, نشاط Artifactual, ونشاط الخلية الكاملة من النوكليوسيد الريبوهيدرولاسيس

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...