Name: quantification of hypopigmentation activity in vitro
Uploaded: 2019-03-06T12:30:00
Description: Watch this Scientific Journal Video about Quantification of Hypopigmentation Activity In Vitro at JoVE.com

Dette arbejde blev støttet af Korea Institut for planlægning og evaluering for teknologi inden for fødevarer, landbrug og skovbrug (IPET) gennem programmet Agri-bioindustrien teknologi udvikling, finansieret af Ministeriet for landbrug, fødevarer og landdistrikter anliggender (MAFRA ) (116159-02-2-WT011) og skole af biovidenskab og bioteknologi i BK21 PLUS, Korea Universitet.

1. forberedelse af Medium, stoffer og reagenser
<ol><li>Forberede B16F10 komplet vækstmediet. Supplement Dulbecco ændrede Eagle's medium (DMEM) med 10% føtal bovint serum (FBS) og 1% penicillin/streptomycin (PEST).<ol>
<li>For at gøre 500 mL af komplette mellemstore, mix 445 mL Dulbeccos modificerede Eagle medium (DMEM) med 50 mL af FBS og 5 mL af PEST i 1 L steriliseret glasflaske. Efter blandingen forsigtigt, filtreres mediet en 0,2 µm flaske-top filter til en ny steriliseret glasflaske og derefter opbevares ved...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+melanogenesis+and+mechanisms+of+skin-lightening+agents+-+existing+and+new+approaches.">The melanogenesis and mechanisms of skin-lightening agents - existing and new approaches.</a> International Journal of Cosmetic Science. 33 (3), 210-221 (2011).</li><li>Passeron, T., Coelho, S. G., Miyamura, Y., Takahashi, K., Hearing, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+and+in+situ+hybridization+in+the+study+of+human+skin+melanocytes.">Immunohistochemistry and in situ hybridization in the study of human skin melanocytes.</a> Experimental Dermatology. 16 (3), 162-170 (2007).</li><li>Lee, J. H., 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=Momilactione+B+inhibits+protein+kinase+A+signaling+and+reduces+tyrosinase-related+proteins+1+and+2+expression+in+melanocytes.">Momilactione B inhibits protein kinase A signaling and reduces tyrosinase-related proteins 1 and 2 expression in melanocytes.</a> Biotechnology Letters. 34 (5), 805-812 (2012).</li><li>Jun, H. J., 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=Dual+inhibitions+of+lemon+balm+(Melissa+officinalis)+ethanolic+extract+on+melanogenesis+in+B16-F1+murine+melanocytes:+inhibition+of+tyrosinase+activity+and+its+gene+expression.">Dual inhibitions of lemon balm (Melissa officinalis) ethanolic extract on melanogenesis in B16-F1 murine melanocytes: inhibition of tyrosinase activity and its gene expression.</a> Food Science and Biotechnology. 20 (4), 1051-1059 (2011).</li><li>Jun, H. J., 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=p-Coumaric+acid+inhibition+of+CREB+phosphorylation+reduces+cellular+melanogenesis.">p-Coumaric acid inhibition of CREB phosphorylation reduces cellular melanogenesis.</a> European Food Research and Technology. 235 (6), 1207-1211 (2012).</li><li>Jun, H. J., 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=Dual+inhibition+of+&#947;-oryzanol+on+cellular+melanogenesis:+inhibition+of+tyrosinase+activity+and+reduction+of+melanogenic+gene+expression+by+a+protein+kinase+a-dependent+mechanism.">Dual inhibition of &#947;-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase a-dependent mechanism.</a> Journal of Natural Products. 75 (10), 1706-1711 (2012).</li><li>Choi, Y. M., 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=Effects+of+the+isoflavone+puerarin+and+its+glycosides+on+melanogenesis+in+B16+melanocytes.">Effects of the isoflavone puerarin and its glycosides on melanogenesis in B16 melanocytes.</a> European Food Research and Technology. 231 (1), 75-83 (2010).</li><li>Cho, B. R., Jun, H. J., Thach, T. T., Wu, C., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Betaine+reduces+cellular+melanin+content+via+suppression+of+microphthalmia-associated+transcription+factor+in+B16-F1+murine+melanocytes.">Betaine reduces cellular melanin content via suppression of microphthalmia-associated transcription factor in B16-F1 murine melanocytes.</a> Food Science and Biotechnology. 26 (5), 1391-1397 (2017).</li><li>Overwijk, W. W., Restifo, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B16+as+a+mouse+model+for+human+melanoma.">B16 as a mouse model for human melanoma.</a> Current Protocols in Immunology. , (2001).</li><li>Virador, V. M., Kobayashi, N., Matsunaga, J., Hearing, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+protocol+for+assessing+regulators+of+pigmentation.">A standardized protocol for assessing regulators of pigmentation.</a> Analytical Biochemistry. 270 (2), 207-219 (1999).</li><li>D'Mello, S. A. N., Finlay, G. J., Baguley, B. C., Askarian-Amiri, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+pathways+in+melanogenesis.">Signaling pathways in melanogenesis.</a> International Journal of Molecular Sciences. 17 (7), 1144 (2016).</li><li>Hou, L., Panthier, J. J., Arnheiter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+and+transcriptional+regulation+in+the+neural+crest-derived+melanocyte+lineage:+interactions+between+KIT+and+MITF.">Signaling and transcriptional regulation in the neural crest-derived melanocyte lineage: interactions between KIT and MITF.</a> Development. 127 (24), 5379-5389 (2000).</li><li>Hedley, S. J., Gawkrodger, D. J., Weetman, A. P., MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;-MSH+and+melanogenesis+in+normal+human+adult+melanocytes.">&#945;-MSH and melanogenesis in normal human adult melanocytes.</a> Pigment Cell Research. 11 (1), 45-56 (1998).</li><li>Hu, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+evaluation+of+melanin+content+and+production+of+pigment+cells+in+vitro.">Methodology for evaluation of melanin content and production of pigment cells in vitro.</a> Photochemistry and Photobiology. 84 (3), 645-649 (2008).</li><li>Carriel, V. 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Vi præsenterede protokoller for at vurdere hypopigmentering aktiviteten af test forbindelser ved hjælp af kulturperler melanocytter. De repræsentative resultater viste hypopigmentering effekt af arbutin, en tyrosinase hæmmer, der hæmmede tyrosinase aktivitet og cellulære melanin indhold. Disse metoder er meget udbredt i anti-melanogenic aktivitet forskning. Bruger disse assays, vi har også med succes identificeret flere bioaktive stoffer, der har melanogenesis hæmmende effekter på B16F10 celler i sidste ti år<s...

Melanin spiller en afgørende rolle i fysiologi, patologi og toksikologi af flere organer, herunder hud, øjne og hjerne1. Hovedfunktioner af melanin er foto-screening og biokemiske virkninger. Melanin absorberer nær-infrarødt og synligt lys samt ultraviolet (UV) stråling, med øget absorption satser ved kortere bølgelængder af lys; således beskytter melanin væv fra skader forårsaget af synligt lys eller UV stråling2. Melanin er en antioxidant og har en affinitet for metaller og andre giftige kemikalier; Derfor kan det beskytte væv fra oxidative og kemiske stress3. Dog, overdreven produktion af melanin forårsager dermatologiske problemer.
Mængden og kvaliteten af melanin i huden og iris er de vigtigste determinanter for farven på iris og hud. Enkeltpersoner kan have forskellige hud farvepræferencer; nogle fordel garvet hud, mens andre favor lysere hudfarver. Afhængigt af disse forbruger profiler, er hypopigmentering kosmetik blevet udviklet for at opfylde individuelle markeder for begunstiget hud farver4. Derfor er undersøgelser af hypopigmentering og anti-melanogenic aktivitet vigtigt både videnskabeligt og praktisk.
Melanogenesis er en kompleks proces af melanin biosyntesen gennem en serie af enzymatiske og spontane kemiske reaktioner i melanocytter. Én melanocyte er omgivet af ca 36 keratinocytter og melanocytter er melanin syntese fabrikker, som distribuerer deres produkt til tilstødende keratinocytter. I huden transporteres melanin produceret og gemt i melanosomal rum af melanocytter til tilstødende keratinocytter i epidermis via dendritter.
L-tyrosin tjener som den oprindelige substrat for melanogenesis og enzymet tyrosinase katalyserer to på hinanden følgende reaktioner, der konverterer L-tyrosin i 3,4-dihydroxyphenylalanine (DOPA) og derefter ind i dopaquinone. Disse reaktioner er det hastighedsbegrænsende trin i melanogenesis5,6. Hypopigmentering aktivitet kan derfor først måles ved vurdering af cellulære tyrosinase aktivitet direkte. For at gøre det, melanocyte ekstrakter som indeholder tyrosinase inkuberes med DOPA og dopaquinone produceret i prøverne kan måles ved spektrofotometri på 475 nm. Værdierne er normaliseret af protein koncentrationer af prøver og stoffer med hypopigmentering aktivitet resulterer i mindre dopaquinone dannelse sammenlignet med kontrol.
For det andet, hypopigmentering aktivitet kan kvantificeres ved direkte måling af melanin i kulturperler melanocytter. Efter behandling af celler med teststoffet, melanin er udvundet alkalisk betingelser og melanin indhold er kvantificeret ved spektrofotometri på 400 nm. En hypopigmentering agent vil resultere i en lavere melanin indhold end kontrol7.
Endelig, hypopigmentering aktivitet kan kvantificeres ved Fontana-Masson melanin farvning og efterfølgende billedanalyse. I Fontana-Masson farvning, melanin granulat reducerer ammoniak-sølv nitrat til en synlig sort metallic tilstand og de sorte områder af celler i mikroskopiske billeder repræsenterer mængden af melanin.
En hypopigmentering agent giver normalt sammenhængende og sammenlignelige resultater med disse tre metoder, som bekræfter, at aktiviteten af stoffet er gyldig. Alternativt kan det være nyttigt at måle udtryk for centrale gener og proteiner i melanogenesis svar på en test-kemikaliet at undersøge hypopigmentering aktivitet. Tyrosinase er tyrosinase-relaterede proteiner (TRP-1) og dopachrome tautomerase (TRP-2) kritiske enzymer i melanogenesis8. Transskription faktor microphthalmia-associerede transskription faktor (MITF) er en master regulator i melanogenesis og kvantificering af dens gen/protein udtryk niveauer eller en promotor aktivitet assay kan også bruges til at vurdere hypopigmentering aktivitet.

<table>
	<tbody>
		<tr>
			<td>3,4-Dihydroxy-l-phenylalanine</td>
			<td>Sigma</td>
			<td>D9628</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution</td>
			<td>Sigma</td>
			<td>#320145</td>
			<td></td>
		</tr>
		<tr>
			<td>Arbutin</td>
			<td>Fluka</td>
			<td>#10960</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>HyClone</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Yakuri Pure Chemicals</td>
			<td>#16223</td>
			<td>37%</td>
		</tr>
		<tr>
			<td>Gold chloride</td>
			<td>American MasterTech</td>
			<td>AHG0226</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Samchun chemical</td>
			<td>H0255</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Bio basic Canada Inc.</td>
			<td>PB0440</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>#60218</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>J.T.Baker</td>
			<td>#3817-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Duksan pure chemicals</td>
			<td>#81</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>655104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear fast red</td>
			<td>Merck</td>
			<td>100121</td>
			<td>Nuclear fast red 0.1% in 5% aluminum sulfate.</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Duksan Pure Chemicals</td>
			<td>#900</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>J.T. Baker</td>
			<td>#3817-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic (Na2HPO4)</td>
			<td>Sigma</td>
			<td>S5011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiosulfate pentahydrate</td>
			<td>Duksan Pure Chemicals</td>
			<td>#2163</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Bio Basic Canada</td>
			<td>TB0196</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Union Carbide</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrosinase from mushroom</td>
			<td>Sigma</td>
			<td>T3824</td>
			<td>25 KU</td>
		</tr>
	</tbody>
</table>

quantification of hypopigmentation activity in vitro

Denne undersøgelse præsenterer laboratoriemetoder til kvantificering af hypopigmentering aktivitet in vitro. Melanin, den store pigment i melanocytter, er syntetiseret i svar til flere cellulære og miljømæssige faktorer. Melanin beskytter hudceller fra ultraviolet skader, men også har biofysiske og biokemiske funktioner. Overdreven produktion eller ophobning af melanin i melanocytter kan forårsage dermatologiske problemer, såsom fregner, mørke pletter, melasma og modermærker. Derfor er kontrol af melanogenesis med hypopigmentering agenter vigtigt hos personer med klinisk eller kosmetiske behov. Melanin er primært syntetiseret i melanosomer af melanocytter i en kompleks biokemiske proces, der kaldes melanogenesis, som er påvirket af ydre og iboende faktorer, såsom hormoner, betændelse, alder og ultraviolet lys eksponering. Vi beskriver tre metoder til at bestemme hypopigmentering aktiviteter kemikalieproducenter eller naturlige stoffer i melanocytter: måling af 1) cellulære tyrosinase aktivitet og 2) melanin indhold og 3) farvning og kvantificere cellulære melanin med billede analyse.
I melanogenesis katalyserer tyrosinase trinnet trafikhastighedsbegrænsning, der konverterer L-tyrosin i 3,4-dihydroxyphenylalanine (L-DOPA) og derefter ind i dopaquinone. Hæmning af tyrosinase er derfor en primær hypopigmentering mekanisme. I kulturperler melanocytter, kan tyrosinase aktivitet kvantificeres ved at tilføje L-DOPA som substrat og måling af dopaquinone produktion af spektrofotometri. Melanogenesis kan også måles ved kvantificere melanin indhold. Melanin-holdige cellulære brøken ekstraheres med NaOH og melanin er kvantificeret spektrofotometrisk. Endelig, melanin indhold kan kvantificeres ved billedanalyse efter Fontana-Masson farvning af melanin. Selv om resultaterne af disse in vitro-assays ikke kan altid gengives i menneskelige hud, er disse metoder almindeligt anvendt i melanogenesis forskning, især som det første skridt til at identificere potentielle hypopigmentering aktivitet. Disse metoder kan også bruges til at vurdere melanocyte aktivitet, vækst og differentiering. Ensartede resultater med de tre forskellige metoder sikrer gyldigheden af virkningerne.

Repræsentative resultater for hypopigmentering aktivitet af arbutin, en anti-melanogenic sammensatte i B16F10 Melanocytter er vist nedenfor. Figur 1A viser, at arbutin betydeligt undertrykt cellulære tyrosinase aktivitet sammenlignet med kontrolelementet køretøjets-behandlet. På samme måde, melanin indhold af celler stimuleret med arbutin blev reduceret betydeligt sammenlignet med kontrol (figur 1B). Mikroskopiske billeder ...

Watch this Scientific Journal Video about Quantification of Hypopigmentation Activity In Vitro at JoVE.com

Quantification of Hypopigmentation Activity In Vitro

Vi beskriver tre eksperimentelle metoder til evaluering af hypopigmentering aktivitet af kemikalier in vitro: kvantificering af 1) cellulære tyrosinase aktivitet og 2) melanin indhold, og 3) måling af melanin af cellulære melanin farvning og billede analyse.

Kvantificering af hypopigmentering aktivitet In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Denne metode kan være nyttige i udviklingen af funktionelle kosmetik og hud agent med anti-melanogene aktiviteter. Det er der er at udføre, og resultatet kan opnås hurtigt. Desuden kan denne metode være nyttig for forskere, der ønsker at identificere eller undersøge virkningerne eller forbindelserne eller undersøge de antimelanogene eller promelanogene aktiviteter.At måle til tyrosinase aktivitet begynder med såning B16-F10 Melanicider på en fem gange 10 til den fjerde celler pr brønd af en 24 godt plade koncentration i komplet medium i 24 timer i en cellekultur inkubator. Den næste dag, erstatte supernatant i hver brønd med DMEM suppleret med frisklavet testforbindelse og inhibitor kontrol eller en negativ kontrol og returnere pladen til rugemaskinen i yderligere 72 timer. Ved afslutningen af behandlingen skylles brøndene to gange med 300 mikroliter kold pbs pr. brønd og læg pladen på is.Næste tilføje 300 mikroliter lysis buffer til hver brønd ved hjælp af en celle skraber til at indsamle cellen lysates efter fem minutter. De resulterende cellepartikelsuspensioner overføres til individuelle 1,5 milliliter mikrocentrifugerør. Og homogenisere lysates tre gange i to sekunder ved 14, 500 rpm på is til at frigive intercellulære tyrosinase.Pellet cellerester ved centrifugering og overfør supernatanterne til individuelle 1,5 milliliter centrifugerør på is. Fordel 70 mikroliter af hver supernatant til individuelle brønde af en klar 96 brønd polystyren mikroplade og tilsæt 140 mikroliter tyrosinase substrat opløsning til hver brønd af supernatant. Ryst pladen blid for at blande brøndens indhold.Derefter inkuberes pladen ved 37 grader celsius i to timer. Og måle til tyrosinase aktivitet på 475 nanometer på en mikroplade læser. At måle melaninindholdet i cellerne efter indsamling af lysatet af de behandlede celler som påvist.Opsaml lysaterne ved centrifugering, og overfør supernatanterne til nye rør. Der tilsættes 300 mikroliter af en normal natriumhydroxid til hver pellet i en times inkubation ved 60 grader Celsius. Efterfulgt af centrifugering af de opløste cellesuspensioner.Derefter overføres 200 mikroliter af supernatanterne til hver brønd på en 96 brøndplade. Og måle melaninindholdet på en mikropladelæser ved en 400 nanometerbølgelængde. For Fontana-Masson Farvning vaskes de behandlede celler to gange med 300 mikroliter kold pbs, og cellerne fastgøres med 200 mikroliter på 10% formalin i en time ved fire grader Celsius.Skyl de tykke celler med destilleret vand og tilsæt 200 mikroliter på 58 til 68 grader celsius ammoniak sølv arbejdsløsning til hver brønd. For en time inkubation ved 37 grader Celsius eller indtil cellerne bliver gul-brun i farven. Skyl de farvede celler med destilleret vand efterfulgt af to minutters inkubation i 0,1% guldchlorid ved stuetemperatur.Skyl guldchloridbehandlede celler med destilleret vand og tilsæt 200 mikroliter natrium thiosulfatopløsning til cellerne. Efter to minutter skylles cellerne igen. Og mærke cellerne med 200 mikroliter af nuklear hurtig rød pr brønd i fem minutter.Vask derefter de farvede celler med vand fra hanen, før der billeddannelse under et let mikroskop. Hvis du vil have tærskelanalyse, skal du åbne billederne i billede J og vælge billede, justere og farvegrænse. For at måle det område, der er bejdset med Fontana Masson, skal du indstille lysstyrkeparameteren til nul og justere lysstyrken til det punkt, hvor alle de sorte eller brune farvede celler er inkluderet.Vælg Analysér og analysér partikler, og markér feltet Sammenfat i vinduet Analysér partikler, og klik derefter på okay for at få en oversigt over resultaterne og kopiere og indsætte dataene i et regneark. Arbutin behandling betydeligt undertrykker cellulære tyrosinase aktivitet i forhold til kontrol køretøj behandlede celler. Tilsvarende melaninindholdet i celler stimuleret med Arbutin er væsentligt reduceret i forhold til kontrollerne.Selv om cellerne er morfologisk ens mellem behandlingsgrupper, Arbutin nedsætter området af sort pigment i forhold til kontrolbehandlede celler. Denne metode er nyttig til aktivitetsscreening ved hjælp af vores sammensatte bibliotek. Der er over hundredvis af prøver, der skal testes hurtigt.Desuden kan denne metode også bruges til at undersøge mekanismen, der involverer melanogenese. Efter de bioaktive kandidater forbindelser er blevet identificeret, for deres vidnesbyrd om studiet af biologiske mekanismer eller kommercielle anvendelser.

quantification-of-hypopigmentation-activity-in-vitro

Research

JoVE Journal

Biochemistry

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Påvisning af pH-afhængige aktivitet af<em&gt; Escherichia coli</em&gt; Chaperone HdeB<em&gt; In vitro</em&gt; og<em&gt; In vivo</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

Kvantificering af overfloden og opladning niveauer af overførsel RNA'er i Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

Kortlægning stofskifte: Overvågning af laktat Dehydrogenase aktiviteten direkte i væv

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

En In Vitro Assay at opdage tRNA-Isopentenyl-Transferase aktivitet

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Kalibrering-fri In Vitro kvantificering af Protein Homo-oligomerisering ved hjælp af kommercielle instrumentering og gratis, Open Source lysstyrke analyse Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

En kolorimetrisk analyse af citrat syntase aktivitet i Drosophila melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. In C. elegans, 2-AG has been found to possess different roles, including modulation of dauer formation and cholesterol metabolism. This report describes a method to overcome the difficulties associated with the costs and stability of deuterated standards required for 2-AG quantification. The procedure for the synthesis of the standard is simple and can be performed in any laboratory, without the need for organic synthesis expertise or special equipment. In addition, a modification of Folch&#39;s method to extract the deuterated standard from C. elegans culture is described. Finally, a quantitative and analytic method to detect 2-AG using the stable isotopically labeled analog 1-AG-d5 is described, which provides reliable results in a fast-chromatographic run. The procedure is useful for studying the multiple roles of 2-AG in C. elegans while also being applicable to other studies of metabolites in different organisms.

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work describes a robust and straightforward method to detect and quantify the endocannabinoid 2-arachidonoylglycerol (2-AG) in C. elegans. An analytical deuterated standard us prepared and used for the quantification of 2-AG by isotopic dilution and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

Syntese af en deutereret standard for kvantificering af 2-Arachidonoylglycerol i Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Kvantificering af protein interaktions netværk dynamik ved hjælp af Multiplexed Co-Immunopræcipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

Kvantificering af coenzym A i celler og væv

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

Kvantificering af syntese og distribution af mtDNA med høj kapacitet

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Forfatter Spotlight: Billedbaseret kvantificering af mitokondriel DNA-syntese og -fordeling med høj kapacitet  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

Billedbaseret kvantificering med høj kapacitet af mitokondrie-DNA-syntese og -distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...