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.

הערה: טחולים נגזרו מעכברים (6-10 שבועות של גיל) וכל ניסויים בבעלי החיים בוצעו בהתאם להנחיות האתיקה של בעלי החיים של פיטר MacCallum מרכז הסרטן (E486). 1. הכנת דוגמאות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנת תאי NK העכבר מופעלים. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בודד תאי NK נאיביים עיקריים מתא בודד השעיות של הטחולים של C57BL / 6 או B6.GrzmB - / - עכברים (null גן GzmB) על ידי סלקציה שלילית באמצעות ערכות זמינות מסחרי. </li><li style=";text-align:right;direction:rtl"> עקוב פרוטוקול של היצרנים. בקצרה, מגנטי תווית תאים &quot;הלא NK&quot; כמו T ותאי B, תאים דנדריטיים, גרנולוציטים, מקרופאגים, תאי דם אדומים באמצעות נוגדני ביוטין מצומדות- נגד סמני המשטח הספציפיים שלהם. השתמש בהפרדה מגנטית עם חרוזים אנטי-ביוטין לרוקן תאים &quot;הלא NK&quot;. </li><li style=";text-align:right;direction:rtl"> תרבות מבודדת תאי NK 5-7 ימים במדיה המכילה IL-2 (1000 U / ml) ב700,000 תאים / מיליליטר בצלחות 24 גםב 37 &#160; מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> לחלופין, שימוש הופעל תאי T 19, CTL המוגבל אנטיגן העיקרי מעכברים מהונדסים 20 קולט תא ההארכה הזמן 1 T, CTL שנוצרו בתרבויות מעורבות הלימפוציטים 21, או supernatants מתאי NK מופעלים כדי לקבוע את הפעילות של GzmB המופרש 19. </li></ol></li><li style=";text-align:right;direction:rtl"> תחזוקה של קו אדם NK תא ובידוד של תאי NK עיקריים. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שמור לוקמיה NK (תאי יט) בRoswell Park זיכרון מכון בינוני (RPMI) בתוספת 10% בסרום עגל עוברי. לחלופין, לטהר את תאי NK עיקריים מהדם ההיקפי של נבדקים בריאים ולעורר במשך 2-3 ימים במדיום המכיל IL-2 ב 100 U / ml כפי שתואר קודם לכן 22. </li></ol></li><li style=";text-align:right;direction:rtl"> דור של lysates תא. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שטפו תאים שלוש פעמים PBS (pH 7.4), ואז להשעות במאגר תמוגה P-40 Nonidet (0.1% NP-40, 250 מ&quot;מ NaCl, 25 מ&quot;מ Hepes, 2.5 mM EDTA). באופן אידיאלי, lyse מינימום של 5 x 10 6 </sעד&gt; תאי NK, או 1 x 10 7 תאים T ב 100 חיץ μl. אל תוסיפו מעכבי פרוטאז, כפי שרבים הם מעכבים בלתי הפיכים של פרוטאזות סרין, ובtryptases מסוים כגון GzmA. דגירה על קרח למשך 20 דקות, ולאחר מכן גלולה את הגרעינים בmicrocentrifuge ב 15,000 XG במשך 10 דקות. זורק את הכדור הגרעיני ולהעביר את supernatant לצינור טרי. </li></ol></li><li style=";text-align:right;direction:rtl"> קבע את ריכוז החלבון של lysates. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> משתמש בפרוטוקול זמין מסחרי של בחירה כגון, ברדפורד או חומצת bicinchoninic (BCA) ולקבוע את ריכוז החלבון. ודא שהריכוז הוא ~ 2 מ&quot;ג מינימום / מיליליטר. lysates החנות ב -20 ° C עד נדרשת (פעילות granzyme יציב במשך חודשים רבים ב -20 ° C). </li></ol></li></ol> 2. Granzyme B פעילות Assay <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנת ריאגנטים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן מלאי מ&quot;מ 10 של מצעים (בוק-AAD-S-Bzl או AC-IEPD-PNA) בDMSO (~ 5 מ&quot;ג / מיליליטר) ולאחסן בaliquots ב -20 &amp; #176; C. הכן דילול עובד טרי וזורקים אחרי כל יום, כמו הבוק-AAD-S-Bzl hydrolyzes באופן חלקי על האחסון בDMSO. </li><li style=";text-align:right;direction:rtl"> הכן מלאי 250 מ&quot;מ של מגיב colorimetric (5,5&#39;-dithio-bis (חומצת 2-nitrobenzoic), DTNB) בDMSO (0.09 גר &#39;/ מיליליטר) ולאחסן ב -20 ° C. הכן דילול עבודה טרי בכל יום. מגיב זה נדרש רק למצעים עם קבוצות SBzl מחוון, לא הרשות הפלסטינית. </li><li style=";text-align:right;direction:rtl"> מרכיבים את החיץ טרי (0.1M HEPES, 0.05 M MgCl 2, pH 7.3) בכל 2-3 שבועות. </li></ol></li><li style=";text-align:right;direction:rtl"> להביא DTNB ומצעים סינטטיים לטמפרטורת חדר. מצעים לדלל (01:16, למשל ב1.6 מיליליטר של חיץ 100 μl) וDTNB (1: 250, לדוגמא 10 μl ב2.5 מיליליטר חיץ) ומערבבים היטב. </li><li style=";text-align:right;direction:rtl"> לדלל מינימום של 100 מיקרוגרם lysate חלבון של כל דגימה במאגר לנפח סופי של 160 μl. בעזרת פיפטה רבה להוסיף 50 μl לכל גם בשלושה עותקים (~ 33.3 מיקרוגרם / טוב) ב&#39;&#39;U&#39; &#39;צלחת 96-היטב ויניל התחתון.כולל &quot;חיץ בלבד&quot; שליטה (גם בשלושה עותקים). </li><li style=";text-align:right;direction:rtl"> הוספה של DTNB המדולל 100 μl לשליטת דגימות / חיץ (להשמיט את הצעד הזה בעבודה עם Ac-IEPD-PNA. הערה: מחשוף של Ac-IEPD-ידי הרשות הפלסטינית GzmB ישירות משחרר fluorogenic PNA (איור 4). </li><li style=";text-align:right;direction:rtl"> השתמש בפיפטה רבה כדי להוסיף במהירות 50 μl של מצעים מדוללים לכל סט של triplicates, החל במאגר אז כל בקרות שליליות וסיים עם דגימות חיוביות צפויות. </li><li style=";text-align:right;direction:rtl"> מניחים את הצלחת בקורא הצלחת. למדוד את פעילות Asp-ASE ידי הידרוליזה של בוק-עלא-עלא-Asp-S-Bzl / Ac-IEPD-הרשות הפלסטינית בקורא microplate בננומטר OD 405. משתמש בפרוטוקול assay הקינטית, לקחת 25 קריאות, בכל 15 שניות (זמן קריאה כוללת 6 דקות בערך). </li><li style=";text-align:right;direction:rtl"> השתמש בקריאות ספיגה שנוצרה בכל נקודת זמן כדי לחשב באופן ידני את שיעור מחשוף מצע אם התוכנה של הצלחת-הקורא לא יכולה ליצור ערך עבור Vmax. </li></ol> 3. ניתוח של Data הערה: הנתונים המופקים מוצג כמהירות המרבית, מוגדר כשינוי של OD לאורך זמן (mod / min). <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי לנתח את הנתונים, לקבוע את השיעור המרבי של מחשוף מצע, המחושב משיעור השינוי בספיגה. לעשות את זה באופן אוטומטי באמצעות תוכנת צלחת קורא, אשר בדרך כלל יש באפשרות זו. </li><li style=";text-align:right;direction:rtl"> לחלופין לאחר צפייה בחלקות הקינטית של שינוי בספיגה, אשר נוצר במהלך assay הקינטית על קורא הצלחת, בחר ידני נקודות נתונים שנותנים הקו הישר התלול ביותר. עבור רוב מבחני השיעור המרבי מושגת בתוך 2 דקות הראשונות של assay. </li><li style=";text-align:right;direction:rtl"> לחסר קריאת OD הראשונית מקריאת OD הגבוהה ביותר בקו הזה ישר ולחלק את מרווח הזמן. העבר את הנתונים גולמיים לכרית Microsoft Excel / גרף לניתוח סטטיסטי ותצוגה גרפית. </li></ol>

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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunodetection+of+granzyme+B+tissue+distribution+and+cellular+localisation.">Immunodetection of granzyme B tissue distribution and cellular localisation.</a> Methods Mol Biol. 844, 237-250 (2012).</li><li>Jenkins, M. 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=Visualizing+CTL+activity+for+different+CD8++effector+T+cells+supports+the+idea+that+lower+TCR/epitope+avidity+may+be+advantageous+for+target+cell+killing.">Visualizing CTL activity for different CD8+ effector T cells supports the idea that lower TCR/epitope avidity may be advantageous for target cell killing.</a> Cell Death Differ. 16 (4), 537-542 (2009).</li><li>Edwards, K. M., Kam, C. M., Powers, J. C., Trapani, J. 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.

מבחינה הסטורית granzymes זוהו כמולקולות מפעיל עיקריות של לימפוציטים מסוג רעיל לתאים (תאי NK וCTL) מסוגל גרימת מוות אפופטוטיים מהיר בתאי מטרה. זה היה בעיקר בגלל הפעולה של GzmB, שהבקיעה מולקולות מצע היעד בaspartate שאריות (ד &#39;) ובכך הייתה מסוגל להפעיל את מפל caspase על ידי שני פרו-caspases ?...

Granzymes היא משפחה של סרין פרוטאזות נמצאה בlysosomes ההפרשה של רוצח טבעי (NK) תאים ולימפוציטים מסוג T ציטוטוקסי (CTL) 1. חמש granzymes שונה קיימים בבני אדם (A, B, H, K ו- M), ועשרה בעכברים (- G, K, M ו- N) 2,3. Granzyme וGranzyme B (GzmA, GzmB) הם הנפוצים ביותר ונחקר בהרחבה בהגדרה האנושית ומכרסמת. הפונקציה הקלאסית של GzmB היא האינדוקציה של אפופטוזיס בתאי מטרת להורג בשיתוף עם perforin חלבון יוצרים הנקבובית, המאפשר לgranzyme לגשת cytosol תא המטרה 4. למרות שביטוי GzmB נמצא באופן חד משמעי בלימפוציטים מסוג רעיל לתאים, מחקרים שנעשו לאחרונה שטיפול במגוון של סוגי תאים להביע GzmB אחרים, לרבות אך לא רק לקרטינוציטים 5, בזופילים 6, 7 תאי פיטום, תאים דנדריטיים plasmacytoid 8, ותאי B 9, 10.בהקשר זה, פונקציות GzmB אינן אפופטוטיים נחשפו החל מההשתתפות בתהליכים דלקתיים, שיפוץ רקמות ומאפיינים אחרים immunoregulatory 11-14. בהתחשב בכך שתפקיד ביולוגי רחב יותר הוצע לGzmB מאשר חשדו בעבר, חוקרים דורשים כלים אמינות וספציפיים לגילויה. יתרון הוא הדרישה הספציפית של GzmB לדבוק בצד carboxyl של שאריות חומצה אספרטית, נכס ייחודיים בין פרוטאזות סרין אוקריוטים 15. עכבר, אנושי וGzmB החולדה הם מבני דומים מאוד, עם זאת סגוליות המצע המורחב של GzmB העכבר שונה בעדינות מזה של שני בני האדם והעכברים 16, מה שאומר שמצעים מסוימים גנריות עם ASP במסוף (P1) ניתן ביקעו ביעילות על ידי GzmB מכל שלושת המינים, ואילו מצעים אחרים עם רצפים מורכבים יותר במעלה הזרם של P1 עשוי לתת תוצאות משתנים באופן נרחב. בעבר ובli האחרון יותרterature, עובדה זו גרמה לבלבול ופירוש לא נכון של המשמעות הביולוגית של כמה ממצאים ניסיוניים רבים, אף על פי שמבוקרת בקפידה, מחקרים הקינטית ביקשו לתקן את המצב 17. במאמר זה אנו בקשנו להמחיש נקודות אלה באמצעות שני מצעים זמינים מסחרי, כלומר בוק-עלא-עלא-Asp-SBzl וN-אצטיל-איל-Asp-p-nitroanilide Glu-Pro. שני ריאגנטים לעשות ליצור קבוצות תגובתי שונות הבאות מחשוף (sulphydryl חופשי מול paranitroanilide חופשי ניאון), אבל אין לכך שום השפעה על מה שמחשוף פרוטאוליטים. הפרוטוקול המתואר הוא adaption מודרנית של פרוטוקול ישן מאוד 18, אבל צריך לעזור לחוקרים להשתמש במצעי GzmB השונים כראוי, גם בעת מתן מסגרת המתודולוגית לאיתור הפעילות של granzymes האחר, כגון GzmA וGzmH.

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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<td>Biorad iMark</td>
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			<td>Biorad Microplate Manager Software Version MPM6.3</td>
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			<td height="21" style="height:21px;">Serocluster U-bottom vinyl 96-well plate</td>
			<td>Corning, MA, USA</td>
			<td>2797</td>
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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.

מצע הבוק-AAD-S-Bzl הוא ספציפי לGzmB GzmB פרוטאז סרין הוא מרכיב עיקרי של לימפוציטים ציטוטוקסיות (תאי CTL וNK) והוא בעיקר אחראי לגרימת מוות אפופטוטיים מהיר בתאי מטרה, כגון תאים או הפך נגוע בנגיף. זה נובע במידה רבה העדפת המצע שלה למחשו?...

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.

Colorimetric Assay שמודד את הפעילות באופן ספציפי Granzyme B מפרקי חלבונים: הידרוליזה של הבוק-עלא-עלא-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 ...

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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.

מבחני colorimetric מהירים לאופן איכותי להבחין RNA ו-DNA בדגימות biomolecular

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.

מדידת מצב יציב, טרום מצב יציב, וחד מחזור קינטי לפעילות ה-DNA 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.

ניטור קרינה מבוססת פעילות PAD4 באמצעות אנלוגי מצע פרו-הקרינה

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.

מדידת אלקטרוכימיות הדינמית של יוני כלוריד

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.

שיטה למניפולציות מתח פנים של מתכת נוזלית באמצעות חמצון פני שטח והפחתה

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.

הכנה תגובתיות של מלח ברומיד Triphosphenium: מקור נוח ויציב של זרחן (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.

המדידה בו זמנית HDAC1 ופעילות HDAC6 הלה תאים באמצעות 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 ...

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.

Microfluidic הכנת Elastomer גבישי נוזלי מפעילים

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.

סינתזה של אסטרים ויה Esterification Steglich ירוק יותר ב- Acetonitrile

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-מבוסס על פעילות מבוססת על קביעת עיכוב מורכבים, IC50 ערכים, פעילות פרועובדתית, ואת הפעילות התא כולו של נוקלאוטילסייד הריבוליטיות

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