Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

University of Tennessee

&#x89D2;&#x819C;&#x548C;&#x6BDB;&#x56CA;&#x7684;&#x7A33;&#x6001;&#x53D6;&#x51B3;&#x4E8E;&#x4EBA;&#x53E3;&#x672C;&#x5730;&#x5316;&#x5728;&#x7279;&#x6B8A;&#x9886;&#x57DF;&#x4E2D;&#x7684;&#x89D2;&#x86CB;&#x767D; (K) 14 &#x8868;&#x793A;&#x5E72;&#x7EC6;&#x80DE;&#x6240;&#x4EA7;&#x751F;&#x7684;&#x5B50;&#x4EE3;&#x7EC6;&#x80DE;&#x4E0D;&#x65AD;&#x4F9B;&#x5E94;&#x3002;&#x82E5;&#x8981;&#x8C03;&#x67E5;&#x5408;&#x79DF;&#x7684;&#x6797;&#x57DF; (Clims) &#x5728;&#x4E0A;&#x76AE;&#x7EC4;&#x7EC7;&#x4E2D;&#x7684;&#x6F5C;&#x5728;&#x4F5C;&#x7528;&#xFF0C;&#x6211;&#x4EEC;&#x751F;&#x6210;&#x8F6C;&#x57FA;&#x56E0;&#x5C0F;&#x9F20; K14 &#x542F;&#x52A8;&#x5B50;&#x63A7;&#x5236;&#x4E0B;&#x7684;&#x663E;&#x6027;&#x8D1F;&#x4E2D;&#x5206;&#x5B50; &#xFF08;DN-&#x4E2D;&#x8F7B;&#x673A;&#xFF09;&#x3002;&#x4E0D;&#x51FA;&#x6240;&#x6599;&#xFF0C;K14 &#x63A8;&#x9500;&#x5546;&#x5B9A;&#x5411;&#x89D2;&#x819C;&#x548C;&#x8868;&#x76AE;&#xFF0C;basal cells&#xFF0C;&#x4EE5;&#x53CA;&#x6BDB;&#x56CA;&#x5916;&#x6839;&#x9798;&#x8F6C;&#x57FA;&#x56E0;&#x7684;&#x9AD8;&#x6C34;&#x5E73;&#x8868;&#x8FBE;&#x3002;&#x5728;&#x89D2;&#x819C;&#x4E0A;&#x76AE;&#x7EC6;&#x80DE;&#x3001; &#x8F6C;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x5BFC;&#x81F4;&#x51CF;&#x5C11;&#x7684;&#x8868;&#x8FBE;&#x7C98;&#x9644;&#x5206;&#x5B50; BP180 &#x548C;&#x6709;&#x7F3A;&#x9677;&#x7684; hemidesmosomes&#xFF0C;&#x5BFC;&#x81F4;&#x89D2;&#x819C;&#x4E0A;&#x76AE;&#x8131;&#x79BB;&#x57FA;&#x7840;&#x7684;&#x57FA;&#x8D28;&#x4E0A;&#xFF0C;&#x5176;&#x4E2D;&#x5728;&#x8F6C;&#x539F;&#x56E0;&#x8D77;&#x75B1;&#xFF0C;&#x4ECE;&#x4F24;&#x53E3;&#x548C;&#x708E;&#x75C7;&#x53CD;&#x5E94;&#x3002;&#x7ECF;&#x8FC7;&#x4E00;&#x6BB5;&#x7684;&#x4E0A;&#x76AE;&#x53D8;&#x8584;&#xFF0C;&#x89D2;&#x819C;&#x4E0A;&#x76AE;&#x7EC6;&#x80DE;&#x7ECF;&#x8FC7;&#x5B9A;&#x5411;&#x8BF1;&#x5BFC;&#x5206;&#x5316;&#x4E3A;&#x8868;&#x76AE;&#x6837;&#x7ED3;&#x6784;&#x3002;K14-DN-&#x4E2D;&#x8F7B;&#x673A;&#x5C0F;&#x9F20;&#x4E5F;&#x53D1;&#x5C55;&#x4E0D;&#x80FD;&#x751F;&#x6210;&#x5934;&#x53D1;&#x8F74;&#x7684;&#x529F;&#x80FD;&#x5931;&#x8C03;&#x6027;&#x6BDB;&#x56CA;&#x9010;&#x6E10;&#x5934;&#x53D1;&#x635F;&#x5931;&#x3002;&#x6BDB;&#x56CA;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x6570;&#x91CF;&#x88AB;&#x4E0B;&#x8DCC; K14-DN-&#x4E2D;&#x8F7B;&#x673A;&#x5C0F;&#x9F20;&#xFF0C;&#x6307;&#x793A; Clims &#x6240;&#x9700;&#x7684;&#x6BDB;&#x56CA;&#x5E72;&#x7EC6;&#x80DE;&#x7EF4;&#x6301;&#x81F3;&#x5C11; 60%&#x3002;&#x6B64;&#x5916;&#xFF0C;Clim2 &#x4E0E; Lhx2 &#x5728;&#x4F53;&#x5185;&#xFF0C;&#x8868;&#x660E; Clim2 LIM homeodomain &#x56E0;&#x5B50; Lhx2&#xFF0C;&#x4EE5;&#x524D;&#x663E;&#x793A;&#x5728;&#x6BDB;&#x56CA;&#x5E72;&#x7EC6;&#x80DE;&#x7EF4;&#x4FEE;&#x4E2D;&#x53D1;&#x6325;&#x4F5C;&#x7528;&#x7684;&#x57FA;&#x672C;&#x7684;&#x534F;&#x56E0;&#x6570;&#x8FDB;&#x884C;&#x4EA4;&#x4E92;&#x3002;&#x5728;&#x4E00;&#x8D77;&#xFF0C;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x8868;&#x660E;&#x4E2D;&#x86CB;&#x767D;&#x8D28;&#x5728;&#x89D2;&#x819C;&#x4E0A;&#x76AE;&#x7EC6;&#x80DE;&#x548C;&#x6BDB;&#x56CA;&#x7684;&#x52A8;&#x6001;&#x5E73;&#x8861;&#x4E2D;&#x53D1;&#x6325;&#x91CD;&#x8981;&#x7684;&#x4F5C;&#x7528;&#x3002;

&#x5408;&#x79DF;&#x7684;&#x6797;&#x57DF; &#xFF08;Clims/&#x903B;&#x8F91;&#x8BBE;&#x8BA1;/Nli) &#x89C4;&#x7BA1;&#x89D2;&#x819C;&#x7A33;&#x6001;&#x53CA;&#x7EF4;&#x4FEE;&#x7684;&#x6BDB;&#x56CA;&#x5E72;&#x7EC6;&#x80DE;&#x3002;

The homeostasis of both cornea and hair follicles depends on a constant supply of progeny cells produced by populations of keratin (K) 14-expressing stem cells localized in specific niches. To investigate the potential role of Co-factors of LIM domains (Clims) in epithelial tissues, we generated transgenic mice expressing a dominant-negative Clim molecule (DN-Clim) under the control of the K14 promoter. As expected, the K14 promoter directed high level expression of the transgene to the basal cells of cornea and epidermis, as well as the outer root sheath of hair follicles. In corneal epithelium, the transgene expression causes decreased expression of adhesion molecule BP180 and defective hemidesmosomes, leading to detachment of corneal epithelium from the underlying stroma, which in turn causes blisters, wounds and an inflammatory response. After a period of epithelial thinning, the corneal epithelium undergoes differentiation to an epidermis-like structure. The K14-DN-Clim mice also develop progressive hair loss due to dysfunctional hair follicles that fail to generate hair shafts. The number of hair follicle stem cells is decreased by at least 60% in K14-DN-Clim mice, indicating that Clims are required for hair follicle stem cell maintenance. In addition, Clim2 interacts with Lhx2 in vivo, suggesting that Clim2 is an essential co-factor for the LIM homeodomain factor Lhx2, which was previously shown to play a role in hair follicle stem cell maintenance. Together, these data indicate that Clim proteins play important roles in the homeostasis of corneal epithelium and hair follicles.

Co-factors of LIM domains (Clims/Ldb/Nli) regulate corneal homeostasis and maintenance of hair follicle stem cells.

L&#x26;#39;hom&#xE9;ostasie des deux follicules de cheveux de la corn&#xE9;e et d&#xE9;pend d&#x26;#39;un approvisionnement constant de cellules prog&#xE9;nitrices produites par les populations de la k&#xE9;ratine (K) 14-exprimant les cellules souches localis&#xE9;es dans des niches sp&#xE9;cifiques. Pour &#xE9;tudier le r&#xF4;le potentiel de la co-facteurs de domaines LIM (CLIMS) dans les tissus &#xE9;pith&#xE9;liaux, nous avons g&#xE9;n&#xE9;r&#xE9; des souris transg&#xE9;niques exprimant une mol&#xE9;cule n&#xE9;gative dominante Clim (DN-Clim) sous le contr&#xF4;le du promoteur K14. Comme pr&#xE9;vu, le promoteur K14 dirig&#xE9; niveau &#xE9;lev&#xE9; d&#x26;#39;expression du transg&#xE8;ne dans les cellules basales de la corn&#xE9;e et l&#x26;#39;&#xE9;piderme, ainsi que la gaine externe des follicules pileux. Dans l&#x26;#39;&#xE9;pith&#xE9;lium de la corn&#xE9;e, l&#x26;#39;expression du transg&#xE8;ne provoque diminution de l&#x26;#39;expression des mol&#xE9;cules d&#x26;#39;adh&#xE9;sion et BP180 h&#xE9;midesmosomes d&#xE9;fectueux, ce qui conduit &#xE0; un d&#xE9;collement de l&#x26;#39;&#xE9;pith&#xE9;lium corn&#xE9;en &#xE0; partir du stroma sous-jacent, ce qui provoque des ampoules, des plaies et une r&#xE9;ponse inflammatoire. Apr&#xE8;s une p&#xE9;riode de l&#x26;#39;&#xE9;pith&#xE9;lium amincissement, l&#x26;#39;&#xE9;pith&#xE9;lium de la corn&#xE9;e subit une diff&#xE9;renciation &#xE0; une structure semblable &#xE0; l&#x26;#39;&#xE9;piderme. Les souris K14-DN-Clim &#xE9;galement d&#xE9;velopper la perte de cheveux progressive due aux follicules pileux dysfonctionnels qui ne g&#xE9;n&#xE8;rent tige du cheveu. Le nombre de cellules souches du follicule pileux est diminu&#xE9; d&#x26;#39;au moins 60% en K14-DN-Clim souris, ce qui indique que CLIMS sont requises pour follicule pileux cellules souches d&#x26;#39;entretien. En outre, Clim2 interagit avec Lhx2 in vivo, ce qui sugg&#xE8;re que Clim2 est un co-facteur essentiel pour le facteur de LIM &#xE0; hom&#xE9;odomaine Lhx2, qui a d&#xE9;j&#xE0; &#xE9;t&#xE9; montr&#xE9; &#xE0; jouer un r&#xF4;le dans le follicule des cheveux sur les cellules souches d&#x26;#39;entretien. Ensemble, ces donn&#xE9;es indiquent que les prot&#xE9;ines jouent un r&#xF4;le important Clim dans l&#x26;#39;hom&#xE9;ostasie de l&#x26;#39;&#xE9;pith&#xE9;lium de la corn&#xE9;e et les follicules pileux.

Co-facteurs de domaines LIM (CLIMS / Ldb / NLI) r&#xE9;gulent l&#x26;#39;hom&#xE9;ostasie de la corn&#xE9;e et l&#x26;#39;entretien des cellules souches du follicule pileux

Die Hom&ouml;ostase von Hornhaut und Haarfollikel h&auml;ngt eine konstante Versorgung mit Nachkommenschaft Zellen produziert von Populationen von Keratin (K) 14-Ausdr&uuml;cken Stammzellen in speziellen Nischen lokalisiert. Um die potentielle Rolle von Co-Faktoren von LIM-Domains (Clims) in epithelialen Geweben zu untersuchen, generierten wir transgene M&auml;use ein Dominant-negativen Clim Molek&uuml;l (DN-Clim) unter der Kontrolle des Projekttr&auml;gers K14 zum Ausdruck zu bringen. Wie erwartet, Regie der K14-Projekttr&auml;ger hohe Ebene Ausdruck der Transgene f&uuml;r die Basal cells der Hornhaut und Oberhaut sowie der &auml;u&szlig;eren Wurzelscheide der Haarfollikel. In Hornhaut-Epithel verursacht die Transgenexpression verringerten Ausdruck der Adh&auml;sion Molek&uuml;l BP180 und defekte Dermo, f&uuml;hrt zu Losl&ouml;sung der Hornhaut-Epithel aus den zugrunde liegenden Stroma, die wiederum dazu f&uuml;hrt, dass Blasen, Wunden und eine entz&uuml;ndliche Reaktion. Nach einer Zeit der epithelialen Ausd&uuml;nnung erf&auml;hrt das Hornhaut-Epithel Differenzierung in eine Oberhaut-&auml;hnliche Struktur. Die K14-DN-Clim-M&auml;use entwickeln auch progressive Haarausfall durch dysfunktionale Haarfollikel, die fehlschlagen, Haare zu generieren. Die Anzahl der Stammzellen Haarfollikel wird verringert, um mindestens 60 % in K14-DN-Clim M&auml;use, darauf hinweist, dass Clims f&uuml;r die Haarfollikel Stammzelle Wartung erforderlich sind. Dar&uuml;ber hinaus interagiert Clim2 mit Lhx2 in-vivo, darauf hindeutet, dass Clim2 eine wesentliche Co-factor f&uuml;r den LIM-Hom&ouml;odom&auml;ne-Faktor Lhx2, die zuvor gezeigt wurde, eine Rolle in der Haarfollikel Stammzell-Pflege. Diese Daten zufolge gemeinsam in die Hom&ouml;ostase des Hornhaut-Epithel und Haarfollikel Clim Proteine eine wichtige Rolle spielen.

Co-Faktoren von LIM-Domains (Clims/Ldb/Nli) Regeln Hornhaut Hom&ouml;ostase und Wartung von Stammzellen Haarfollikel.

L&#39;omeostasi di cornea e di follicoli dei capelli dipende da una costante fornitura di celle progenie prodotta da popolazioni di cheratina (K) esprimendo 14 cellule staminali localizzate in specifiche nicchie. Per indagare il ruolo potenziale dei domini di cofattori di LIM (Clims) nei tessuti epiteliali, abbiamo generato topi transgenici che esprimono una molecola di Clim dominante-negativo (DN-Clim) sotto il controllo del promotore K14. Come previsto, il promotore K14 diretto alto livello espressione del transgene per il basal cells della cornea e dell&#39;epidermide, come pure la guaina esterna della radice dei follicoli piliferi. Nell&#39;epitelio cornea, l&#39;espressione del transgene provoca diminuzione espressione della molecola di adesione BP180 ed emidesmosomi difettosi, che portano al distacco dell&#39;epitelio cornea da stroma sottostante, che a sua volta causa vesciche, ferite e una risposta infiammatoria. Dopo un periodo di assottigliamento epiteliale, l&#39;epitelio corneale subisce differenziazione per un&#39;epidermide-come la struttura. I topi K14-DN-Clim sviluppano anche perdita di capelli progressiva a causa disfunzionali follicoli dei capelli che non riescono a generare gli alberi dei capelli. Il numero di cellule staminali capelli follicolo &egrave; diminuito di almeno il 60% nei topi K14-DN-Clim, indicando che Clims sono necessari per la manutenzione delle cellule staminali capelli follicolo. Inoltre, Clim2 interagisce con Lhx2 in vivo, suggerendo che il Clim2 &egrave; un cofattore essenziale per il fattore di homeodomain LIM Lhx2, che precedentemente &egrave; stato indicato per svolgere un ruolo nel mantenimento delle cellule staminali capelli follicolo. Insieme, questi dati indicano che le proteine Clim giocano un ruolo importante nell&#39;omeostasi dell&#39;epitelio corneale e follicoli piliferi.

Cofattori di domini LIM (Clims/Ldb/INL) regolano l&#39;omeostasi cornea e la manutenzione delle cellule staminali del follicolo pilifero.

&#x89D2;&#x819C;&#x3084;&#x6BDB;&#x5305;&#x306E;&#x4E21;&#x65B9;&#x306E;&#x6052;&#x5E38;&#x6027;&#x306F;&#x3001;&#x30B1;&#x30E9;&#x30C1;&#x30F3;&#x306E;&#x500B;&#x4F53;&#x6570;&#xFF08;K&#xFF09;&#x306F;&#x3001;&#x7279;&#x5B9A;&#x306E;&#x30CB;&#x30C3;&#x30C1;&#x306B;&#x5C40;&#x5728;&#x3057;&#x3066;14&#x3092;&#x767A;&#x73FE;&#x3059;&#x308B;&#x5E79;&#x7D30;&#x80DE;&#x306B;&#x3088;&#x3063;&#x3066;&#x7523;&#x751F;&#x3055;&#x5B50;&#x5B6B;&#x7D30;&#x80DE;&#x306E;&#x5B89;&#x5B9A;&#x4F9B;&#x7D66;&#x306B;&#x4F9D;&#x5B58;&#x3057;&#x307E;&#x3059;&#x3002;&#x4E0A;&#x76AE;&#x7D44;&#x7E54;&#x306E;LIM&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#xFF08;Clims&#xFF09;&#x306E;&#x5171;&#x540C;&#x306E;&#x8981;&#x56E0;&#x306E;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x5F79;&#x5272;&#x3092;&#x8ABF;&#x3079;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;&#x6211;&#x3005;&#x306F;K14&#x30D7;&#x30ED;&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x306E;&#x5236;&#x5FA1;&#x4E0B;&#x306B;&#x30C9;&#x30DF;&#x30CA;&#x30F3;&#x30C8;&#x30CD;&#x30AC;&#x30C6;&#x30A3;&#x30D6;&#x306E;Clim&#x5206;&#x5B50;&#xFF08;DN-CLIM&#xFF09;&#x3092;&#x767A;&#x73FE;&#x3059;&#x308B;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30B8;&#x30A7;&#x30CB;&#x30C3;&#x30AF;&#x30DE;&#x30A6;&#x30B9;&#x3092;&#x4F5C;&#x88FD;&#x3057;&#x305F;&#x3002;&#x4E88;&#x60F3;&#x901A;&#x308A;&#x3001;K14&#x30D7;&#x30ED;&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x306F;&#x3001;&#x89D2;&#x819C;&#x3068;&#x8868;&#x76AE;&#x306E;&#x57FA;&#x5E95;&#x7D30;&#x80DE;&#x3068;&#x540C;&#x69D8;&#x306B;&#x3001;&#x6BDB;&#x5305;&#x306E;&#x5916;&#x6BDB;&#x6839;&#x9798;&#x306B;&#x5C0E;&#x5165;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x9AD8;&#x30EC;&#x30D9;&#x30EB;&#x767A;&#x73FE;&#x3092;&#x6307;&#x793A;&#x3057;&#x305F;&#x3002;&#x89D2;&#x819C;&#x4E0A;&#x76AE;&#x3067;&#x306F;&#x3001;&#x5C0E;&#x5165;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x767A;&#x73FE;&#x306F;&#x3001;&#x9806;&#x756A;&#x306B;&#x6C34;&#x3076;&#x304F;&#x308C;&#x3001;&#x50B7;&#x3084;&#x708E;&#x75C7;&#x53CD;&#x5FDC;&#x3092;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3059;&#x57FA;&#x672C;&#x7684;&#x306A;&#x9593;&#x8CEA;&#x304B;&#x3089;&#x3001;&#x89D2;&#x819C;&#x4E0A;&#x76AE;&#x306E;&#x5265;&#x96E2;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B;&#x3001;&#x63A5;&#x7740;&#x5206;&#x5B50;BP180&#x4E0D;&#x826F;&#x30D8;&#x30DF;&#x30C7;&#x30B9;&#x30E2;&#x30BD;&#x30FC;&#x30E0;&#x306E;&#x767A;&#x73FE;&#x4F4E;&#x4E0B;&#x3092;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3057;&#x307E;&#x3059;&#x3002;&#x9593;&#x4F10;&#x4E0A;&#x76AE;&#x306E;&#x671F;&#x9593;&#x306E;&#x5F8C;&#x3001;&#x89D2;&#x819C;&#x4E0A;&#x76AE;&#x306F;&#x8868;&#x76AE;&#x306E;&#x3088;&#x3046;&#x306A;&#x69CB;&#x9020;&#x3078;&#x306E;&#x5206;&#x5316;&#x3092;&#x53D7;&#x3051;&#x308B;&#x3002; K14-DN-&#x306E;Clim&#x30DE;&#x30A6;&#x30B9;&#x306F;&#x307E;&#x305F;&#x3001;&#x6BDB;&#x5E79;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x5931;&#x6557;&#x3057;&#x305F;&#x6A5F;&#x80FD;&#x4E0D;&#x5168;&#x306E;&#x6BDB;&#x5305;&#x306B;&#x8D77;&#x56E0;&#x3059;&#x308B;&#x9032;&#x884C;&#x6027;&#x306E;&#x8131;&#x6BDB;&#x3092;&#x958B;&#x767A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x6BDB;&#x5305;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x6570;&#x304C;Clims&#x306F;&#x3001;&#x6BDB;&#x5305;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x7DAD;&#x6301;&#x306B;&#x5FC5;&#x8981;&#x3068;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3001;K14-DN-&#x306E;Clim&#x30DE;&#x30A6;&#x30B9;&#x3067;&#x306F;&#x5C11;&#x306A;&#x304F;&#x3068;&#x3082;60&#xFF05;&#x6E1B;&#x5C11;&#x3057;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;Clim2&#x306F;Clim2&#x4EE5;&#x524D;&#x306B;&#x6BDB;&#x5305;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x7DAD;&#x6301;&#x306B;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3059;&#x3053;&#x3068;&#x304C;&#x793A;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;LIM&#x30DB;&#x30E1;&#x30AA;&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x56E0;&#x5B50;Lhx2&#x3001;&#x5FC5;&#x9808;&#x306E;&#x88DC;&#x56E0;&#x5B50;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x5506;&#x3057;&#x3001;in vivo&#x3067;Lhx2&#x3068;&#x5BFE;&#x8A71;&#x3057;&#x307E;&#x3059;&#x3002;&#x4E00;&#x7DD2;&#x306B;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x3001;&#x306E;Clim&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306F;&#x3001;&#x89D2;&#x819C;&#x4E0A;&#x76AE;&#x3068;&#x6BDB;&#x5305;&#x306E;&#x6052;&#x5E38;&#x6027;&#x306B;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3059;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

LIM&#x30C9;&#x30E1;&#x30A4;&#x30F3;&#x306E;&#x88DC;&#x56E0;&#x5B50;&#xFF08;Clims / LDB / NLI&#xFF09;&#x304C;&#x6BDB;&#x5305;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x89D2;&#x819C;&#x6052;&#x5E38;&#x6027;&#x3068;&#x30E1;&#x30F3;&#x30C6;&#x30CA;&#x30F3;&#x30B9;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;

&#xAC01; &#xB9C9;&#xACFC; &#xBAA8; &#xB0AD;&#xC758; &#xD56D;&#xC0C1;&#xC131; &#xC790;&#xC190; &#xC138;&#xD3EC; &#xD2B9;&#xC815; &#xD2C8;&#xC0C8;&#xC5D0;&#xC11C; &#xD654; &#xB41C; &#xAC01; &#xC9C8; (K) 14 &#xD45C;&#xD604; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; &#xC778;&#xAD6C;&#xC5D0; &#xC758;&#xD574; &#xC0DD;&#xC0B0;&#xC758; &#xC77C;&#xC815; &#xD55C; &#xACF5;&#xAE09;&#xC5D0; &#xB530;&#xB77C; &#xB2EC;&#xB77C; &#xC9D1;&#xB2C8;&#xB2E4;. &#xC0C1;&#xD53C; &#xC870;&#xC9C1;&#xC5D0; &#xC784; &#xC694;&#xC778;&#xC744; &#xB3C4;&#xBA54;&#xC778; (Clims)&#xC758; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xC5ED;&#xD560; &#xC870;&#xC0AC;&#xB97C; K14 &#xBAA8;&#xD130;&#xC758; &#xC81C;&#xC5B4;&#xC5D0;&#xC11C; &#xC9C0;&#xBC30;&#xC801;&#xC778; &#xBD80;&#xC815;&#xC801;&#xC778; Clim &#xBD84;&#xC790; (DN Clim)&#xB97C; &#xD45C;&#xD604; &#xD558;&#xB294; &#xC720;&#xC804;&#xC790; &#xBCC0;&#xD615; &#xC950;&#xB97C; &#xC0DD;&#xC131; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC608;&#xC0C1; &#xD588;&#xB358; &#xB300;&#xB85C;, K14 &#xBC1C;&#xAE30;&#xC778; basal cells &#xAC01; &#xB9C9;&#xC758; &#xD45C; &#xD53C;, &#xBAA8; &#xB0AD;&#xC758; &#xC678;&#xBD80; &#xB8E8;&#xD2B8; &#xCE7C; &#xC9D1;&#xC5D0; transgene&#xC758; &#xB192;&#xC740; &#xC218;&#xC900;&#xC758; &#xC2DD; &#xAC10;&#xB3C5;. &#xAC01; &#xB9C9; &#xC0C1;&#xD53C;&#xC5D0; transgene &#xC2DD;&#xC774; &#xAC10;&#xC18C; &#xC2DD; &#xC811;&#xCC29; &#xBD84;&#xC790; BP180 &#xBC0F; &#xACB0;&#xD568;&#xC774; &#xC788;&#xB294; hemidesmosomes, &#xC0C1;&#xCC98;&#xC640; &#xC5FC;&#xC99D; &#xBC18;&#xC751;&#xC744; &#xCC28;&#xB840; &#xC6D0;&#xC778;&#xC5D0;&#xB294; &#xBB3C;&#xC9D1;, &#xAE30;&#xBCF8; &#xAE30;&#xC9C8;&#xC5D0;&#xC11C; &#xAC01; &#xB9C9; &#xC0C1;&#xD53C;&#xC758; &#xCD08;&#xC5F0;&#xC744; &#xC120;&#xB3C4; &#xBC1C;&#xC0DD; &#xD569;&#xB2C8;&#xB2E4;. &#xC0C1;&#xD53C; &#xC231;&#xC774;&#xC758; &#xAE30;&#xAC04;, &#xD6C4; &#xAC01; &#xB9C9; &#xC0C1;&#xD53C; &#xD45C; &#xD53C; &#xAD6C;&#xC870;&#xB85C;&#xC5D0; &#xCC28;&#xBCC4;&#xD654;&#xB97C; &#xACAA; &#xC2B5;. K14-DN-Clim &#xC950; &#xBA38;&#xB9AC; &#xC0E4;&#xD504;&#xD2B8; &#xC0DD;&#xC131; &#xC2E4;&#xD328; &#xBD88;&#xD589; &#xB0AD; &#xC9C4;&#xBCF4;&#xC801;&#xC778; &#xD0C8;&#xBAA8;&#xB97C; &#xAC1C;&#xBC1C;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xBA38;&#xB9AC; &#xC5EC; &#xD3EC; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; &#xC218;&#xB294; Clims&#xB294; &#xBA38;&#xB9AC; &#xC5EC; &#xD3EC; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xC720;&#xC9C0;&#xC5D0; &#xD544;&#xC694;&#xD55C; &#xB098;&#xD0C0;&#xB0B4;&#xB294; K14 DN Clim &#xC0DD;&#xC950;&#xC5D0;&#xC11C; &#xC801;&#xC5B4;&#xB3C4; 60% &#xAC10;&#xC18C; &#xB429;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, clim2&#xB294; Lhx2 in vivo Clim2 &#xC784; homeodomain &#xC694;&#xC18C; &#xBA38;&#xB9AC; &#xC5EC; &#xD3EC; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xC720;&#xC9C0; &#xBCF4;&#xC218; &#xC5ED;&#xD560;&#xC744; &#xBCF4;&#xC5EC; &#xC92C;&#xB358; &#xC774;&#xC804;&#xC5D0; lhx2&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC218;&#xC801;&#xC778; co-factor &#xC81C;&#xC548; &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xD569;&#xB2C8;&#xB2E4;. &#xD568;&#xAED8;, &#xC774;&#xB7EC;&#xD55C; &#xB370;&#xC774;&#xD130; Clim &#xB2E8;&#xBC31;&#xC9C8; &#xAC01; &#xB9C9; &#xC0C1;&#xD53C;&#xC640; &#xBAA8; &#xB0AD;&#xC758; &#xD56D;&#xC0C1; &#xC131;&#xC5D0; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xC7AC;&#xC0DD;&#xC744; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;.

&#xC784; &#xB3C4;&#xBA54;&#xC778; (Clims/Ldb/Nli)&#xC758; &#xC694;&#xC778;&#xC744; &#xC870;&#xC808; &#xAC01; &#xB9C9; &#xD56D;&#xC0C1;&#xC131; &#xBC0F; &#xBA38;&#xB9AC; &#xC5EC; &#xD3EC; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; &#xC720;&#xC9C0; &#xBCF4;&#xC218;.

La homeostasis de los dos fol&#xED;culos de c&#xF3;rnea y el cabello depende de un suministro constante de c&#xE9;lulas de la progenie producida por las poblaciones de la queratina (K) 14-que expresan las c&#xE9;lulas madre localizadas en nichos espec&#xED;ficos. Para investigar el papel potencial de la co-factores de dominios LIM (Clims) en los tejidos epiteliales, se generaron ratones transg&#xE9;nicos que expresan una mol&#xE9;cula dominante negativo Clim (DN-Clim) bajo el control del promotor de K14. Como se esperaba, el promotor K14 dirigida alto nivel de expresi&#xF3;n del transg&#xE9;n a las c&#xE9;lulas basales de la c&#xF3;rnea y la epidermis, as&#xED; como la vaina externa de la ra&#xED;z de los fol&#xED;culos pilosos. En epitelio de la c&#xF3;rnea, la expresi&#xF3;n del transg&#xE9;n provoca una disminuci&#xF3;n de expresi&#xF3;n de la mol&#xE9;cula de adhesi&#xF3;n BP180 y hemidesmosomas defectuosos, lo que lleva al desprendimiento del epitelio corneal del estroma subyacente, que a su vez causa ampollas, heridas y una respuesta inflamatoria. Despu&#xE9;s de un per&#xED;odo de adelgazamiento epitelial, el epitelio corneal se somete a la diferenciaci&#xF3;n de una estructura similar a la epidermis. Los ratones K14-DN-Clim tambi&#xE9;n desarrollan la p&#xE9;rdida de cabello progresiva debido a los fol&#xED;culos pilosos disfuncionales que no logran generar los ejes del pelo. El n&#xFA;mero de c&#xE9;lulas madre del fol&#xED;culo piloso se reduce por lo menos 60% en K14-DN-Clim ratones, lo que indica que Clims se requiere para el mantenimiento del fol&#xED;culo piloso de c&#xE9;lulas madre. Adem&#xE1;s, interact&#xFA;a con Clim2 Lhx2 in vivo, lo que sugiere que Clim2 es un co-factor esencial para el LIM homeodominio factor de Lhx2, que se hab&#xED;a demostrado que juegan un papel en el mantenimiento del fol&#xED;culo piloso de c&#xE9;lulas madre. En conjunto, estos datos indican que las prote&#xED;nas Clim desempe&#xF1;an papeles importantes en la homeostasis del epitelio de la c&#xF3;rnea y los fol&#xED;culos pilosos.

Los co-factores de dominios LIM (Clims / BID / INT) regulan la homeostasis de la c&#xF3;rnea y el mantenimiento de las c&#xE9;lulas madre del fol&#xED;culo piloso

Grainyhead transcription factors play an evolutionarily conserved role in regulating epidermal terminal differentiation. One such factor, the mammalian Grainyhead-like epithelial transactivator (Get1/Grhl3), is important for epidermal barrier formation. In addition to a role in barrier formation, Grainyhead genes play roles in closure of several structures such as the mouse neural tube and Drosophila wounds. Consistent with these observations, we found that Get1 knockout mice have an eye-open at birth phenotype. The failure of eyelid closure appears to be due to critical functions of Get1 in promoting F-actin polymerization, filopodia formation, and the cell shape changes that are required for migration of the keratinocytes at the leading edge during eyelid closure. The expression of TGFalpha, a known regulator of leading edge formation, is decreased in the eyelid tip of Get1(-/-) mice. Levels of phospho-EGFR and phospho-ERK are also decreased at the leading edge tip. Furthermore, in an organ culture model, TGFalpha can increase levels of phospho-EGFR and promote cell shape changes as well as leading edge formation in Get1(-/-) eyelids, indicating that in eyelid closure Get1 acts upstream of TGFalpha in the EGFR/ERK pathway.

Grainyhead-like factor Get1/Grhl3 regulates formation of the epidermal leading edge during eyelid closure.

Skin and bladder epithelia form effective permeability barriers through the activation of distinct differentiation gene programs. Using a genome-wide gene-expression study, we identified transcriptional regulators whose expression correlates highly with that of differentiation markers in both the bladder and skin, including the Grainyhead factor Get1/Grhl3, which is already known to be important for epidermal barrier formation. In the bladder, Get1 is most highly expressed in the differentiated umbrella cells and its mutation in mice leads to a defective bladder epithelial barrier formation due to the failure of apical membrane specialization. Genes encoding components of the specialized urothelial membrane, the uroplakins, were downregulated in Get1(-/-) mice. At least one of these genes, uroplakin II, is a direct target of Get1. The urothelial-specific activation of the uroplakin II gene is due to selective binding of Get1 to the uroplakin II promoter in urothelial cells, which is most likely regulated by histone modifications. These results show a crucial role for Get1 in urothelial differentiation and barrier formation.

The epidermal differentiation-associated Grainyhead gene Get1/Grhl3 also regulates urothelial differentiation.

Although homeostasis of rapidly renewing tissues like skin epithelia is maintained by stem cells, the committed progeny of stem cells in the basal layer of epidermis retain regenerative potential and are capable of forming epidermis in response to environmental cues. It is not clear, however, at what point within the epidermal lineage keratinocytes lose this regenerative potential. In this study, we examined the extent of tissue formation by post-mitotic differentiated keratinocytes. We show that cultures of mouse keratinocytes that were, by all measures, differentiated were able to reform a self-renewing, hair-bearing skin when transplanted onto suitable sites in vivo. Genetic labeling and lineage-tracing studies in combination with an involucrin-driven Cre/lox reporter system confirmed that transplanted differentiated keratinocytes were indeed the source of the regenerated skin. More importantly, analysis of early stages of skin regeneration showed hallmarks of dedifferentiation of transplanted differentiated keratinocytes. These data indicate that commitment to differentiation does not prohibit cells from re-entering the cell cycle, de-differentiating, and acquiring "stemness". These findings suggest that epidermis can use different strategies for homeostasis and tissue regeneration.

Regeneration of multilineage skin epithelia by differentiated keratinocytes.

The human growth hormone/chorionic somatomammotropin (hGH/CSH) locus at 17q22-24, consisting of one pituitary-expressed postnatal (GH1) and four placenta-expressed genes (GH2, CSH1, CSH2, and CSHL1), is implicated in regulation of postnatal and intrauterine growth. A positive correlation has been reported between the offspring's birth weight and serum placental GH (coded by GH2) and placental lactogen (coded by CSH1, CSH2) levels in pregnant women.

Differential expression profile of growth hormone/chorionic somatomammotropin genes in placenta of small- and large-for-gestational-age newborns.

The human GH/CSH cluster consisting of one pituitary-expressed (GH1) and four placenta-expressed loci has been implicated in maternal metabolic adaptation to pregnancy, regulation of intrauterine and postnatal growth. We investigated how the mRNA expression profile of placental GH2, CSH1 and CSH2 genes and their alternative transcripts correlates with maternal pre-eclampsia (PE) and/or gestational diabetes mellitus (GD). The expression of studied genes in PE placentas (n=17) compared to controls (n=17) exhibited a trend for reduced transcript levels. The alternative transcripts retaining intron 4, GH2-2 and CSH1-2 showed significantly reduced expression in PE cases without growth restriction (P=0.007, P=0.008, respectively). In maternal GD (n=23), a tendency of differential expression was detected only for the GH2 gene and in pregnancies with large-for-gestational-age newborns. Our results, together with those reported by others, are consistent with a pleiotropic effect of placental hGH/CSH genes at the maternal-fetal interface relating to the regulation of fetal growth and the risk of affected maternal metabolism.

Differential placental expression profile of human Growth Hormone/Chorionic Somatomammotropin genes in pregnancies with pre-eclampsia and gestational diabetes mellitus.

This paper was withdrawn at the request of the editors due to uncertainties inherent in the statistical analysis.

WITHDRAWN: TNF-Related Apoptosis-Inducing Ligand TRAIL as a Potential Biomarker for Early Pregnancy Complications.

Despite the importance of placenta in mediating rapid physiological changes in pregnancy, data on temporal dynamics of placental gene expression are limited. We completed the first transcriptome profiling of human placental gene expression dynamics (GeneChips, Affymetrix®; ~47,000 transcripts) from early to mid-gestation (n = 10; gestational weeks 5-18) and report 154 genes with significant transcriptional changes (ANOVA, FDR P<0.1). TaqMan RT-qPCR analysis (n = 43; gestational weeks 5-41) confirmed a significant (ANOVA and t-test, FDR P<0.05) mid-gestational peak of placental gene expression for BMP5, CCNG2, CDH11, FST, GATM, GPR183, ITGBL1, PLAGL1, SLC16A10 and STC1, followed by sharp decrease in mRNA levels at term (t-test, FDR P<0.05). We hypothesized that normal course of late pregnancy may be affected when genes characteristic to mid-gestation placenta remain highly expressed until term, and analyzed their expression in term placentas from normal and complicated pregnancies [preeclampsia (PE), n = 12; gestational diabetes mellitus (GDM), n = 12; small- and large-for-gestational-age newborns (SGA, LGA), n = 12+12]. STC1 (stanniocalcin 1) exhibited increased mRNA levels in all studied complications, with the most significant effect in PE- and SGA-groups (t-test, FDR P<0.05). In post-partum maternal plasma, the highest STC1 hormone levels (ELISA, n = 129) were found in women who had developed PE and delivered a SGA newborn (median 731 vs 418 pg/ml in controls; ANCOVA, P = 0.00048). Significantly higher expression (t-test, FDR P<0.05) of CCNG2 and LYPD6 accompanied with enhanced immunostaining of the protein was detected in placental sections of PE and GDM cases (n = 15). Our study demonstrates the importance of temporal dynamics of placental transcriptional regulation across three trimesters of gestation. Interestingly, many genes with high expression in mid-gestation placenta have also been implicated in adult complex disease, promoting the discussion on the role of placenta in developmental programming. The discovery of elevated maternal plasma STC1 in pregnancy complications warrants further investigations of its potential as a biomarker.

Jaana Mannik

Department of Biochemistry and Cellular and Molecular Biology

University of Tennessee

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Jaana Mannik

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biochemistry and Cellular and Molecular Biology

University of Tennessee

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Department of Biochemistry and Cellular and Molecular Biology