construction-vapor-chambers-used-to-expose-mice-to-alcohol-during

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

combining-imaging-electrophysiology-to-visualize-record-spreading

Combining Imaging and Electrophysiology to Visualize and Record Spreading Depolarizations in Mice

Spreading Depolarizations (SDs) are massive events in the brain that often go undetected due to their slow propagation through gray matter. Because SD ...

University of New Mexico Health Sciences Center

University of New Mexico School of Medicine

We previously presented evidence that proliferative human islet precursor cells may be derived in vitro from adult islets by epithelial-to-mesenchymal transition (EMT) and show here that similar fibroblast-like cells can be derived from mouse islets. These mouse cell populations exhibited changes in gene expression consistent with EMT. Both C-peptide and insulin mRNAs were undetectable in expanded cultures of mouse islet-derived precursor cells (mIPCs). After expansion, mIPCs could be induced to migrate into clusters and differentiate into hormone-expressing islet-like aggregates. Although early morphological changes suggesting EMT were observed by time-lapse microscopy when green fluorescent protein-labeled beta cells were placed in culture, the expanded precursor cell population was not fluorescent. Using two mouse models in which beta cells were permanently made either to express alkaline phosphatase or to have a deleted M(3) muscarinic receptor, we provide evidence that mIPCs in long term culture are not derived from beta cells.

Endocrine precursor cells from mouse islets are not generated by epithelial-to-mesenchymal transition of mature beta cells.

Islet transplantation offers improved glucose homeostasis in diabetic patients, but transplantation of islets is limited by the supply of donor pancreases. Undifferentiated precursors hold promise for cell therapy because they can expand before differentiation to produce a large supply of functional insulin-producing cells. Previously, we described proliferative populations of human islet-derived precursor cells (hIPCs) from adult islets. To show the differentiation potential of hIPCs, which do not express insulin mRNA after at least 1,000-fold expansion, we generated epithelial cell clusters (ECCs) during 4 days of differentiation in vitro. After transplantation into mice, 22 of 35 ECC preparations differentiated and matured into functional cells that secreted human C-peptide in response to glucose. Transcripts for insulin, glucagon, and somatostatin in recovered ECC grafts increased with time in vivo, reaching levels approximately 1% of those in adult islets. We show that hIPCs are mesenchymal stromal cells (MSCs) that adhere to plastic, express CD73, CD90, and CD105, and can differentiate in vitro into adipocytes, chondrocytes, and osteocytes. Moreover, we find a minor population of CD105(+)/CD73(+)/CD90(+) cells in adult human islets (prior to incubation in vitro) that express insulin mRNA at low levels. We conclude that hIPCs are a specific type of pancreas-derived MSC that are capable of differentiating into hormone-expressing cells. Their ability to mature into functional insulin-secreting cells in vivo identifies them as an important adult precursor or stem cell population that could offer a virtually unlimited supply of human islet-like cells for replacement therapy in type 1 diabetes. Disclosure of potential conflicts of interest is found at the end of this article.

Human islet-derived precursor cells are mesenchymal stromal cells that differentiate and mature to hormone-expressing cells in vivo.

&#x7A33;&#x5B9A;&#x8868;&#x793A;&#x9F20;&#x6807;&#x5305;&#x542B; C &#x7EC8;&#x7AEF;&#x6269;&#x5C55;&#x540D;&#x8D4B;&#x4E88;&#x9AD8;&#x4EB2;&#x548C;&#x6027;&#x7ED3;&#x5408;&#x7684;&#x91D1;&#x73AF;&#x86C7; (alphaBgTx) &#x7684; 5-&#x7F9F;&#x8272;&#x80FA; (3A) Rs &#x66FC;&#x6D66;&#x6C49;&#x514B; 293 &#x7EC6;&#x80DE;&#x7CFB;&#x6210;&#x7ACB; &#xFF08;alphaBgTx-5-HT(3A)Rs) &#x548C;&#x7528;&#x4E8E;&#x51C0;&#x5316; alphaBgTx-5-&#x7F9F;&#x8272;&#x80FA; (3A) Rs &#x4F7F;&#x7528;&#x5149;&#x4EB2;&#x548C;&#x6807;&#x8BB0;&#x7684;&#x7ED3;&#x6784;&#x7814;&#x7A76;&#x4E2D;&#x4F7F;&#x7528;&#x7684;&#x8102;&#x8D28;&#x73AF;&#x5883;&#x4E2D;&#x3002;alphaBgTx-5-&#x7F9F;&#x8272;&#x80FA; (3A) Rs &#x8868;&#x793A;&#x4E86;&#x5F3A;&#x52B2; (BRL 43694 &#x7ED1;&#x5B9A;&#x7AD9;&#x70B9; [H (3)] (&#x5927;&#x7EA6; 3 &#x53D7;&#x4F53; microg) &#x6BCF;&#x6BEB;&#x514B;&#x86CB;&#x767D;&#x7684; 60 pmol) &#x548C;&#x76F8;&#x540C;&#x7684;&#x529F;&#x80FD;&#x5C5E;&#x6027;&#x663E;&#x793A;&#x4E3A;&#x91CE;&#x751F;&#x578B;&#x53D7;&#x4F53; (5-&#x7F9F;&#x8272;&#x80FA; EC(50) = 5.3 + /-0.04 microM&#xFF09;&#x3002;&#x540C;&#x65F6; [(125) i] alphaBgTx &#x7ED1;&#x5B9A;&#x5230; alphaBgTx-5-&#x7F9F;&#x8272;&#x80FA; (3A) Rs &#x9AD8;&#x4EB2;&#x548C;&#x529B; &#xFF08;K(d) = 11 nM&#xFF09;&#xFF0C;&#x975E;&#x653E;&#x5C04;&#x6027; alphaBgTx &#xFF08;&#x6700;&#x591A; 300 microM) &#x7684;&#x5E94;&#x7528;&#x7A0B;&#x5E8F;&#x6709;&#x6CA1;&#x6709;&#x5BF9; 5-&#x7F9F;&#x8272;&#x80FA;&#x8BF1;&#x5BFC;&#x5F53;&#x524D;&#x54CD;&#x5E94;&#x7684;&#x5F71;&#x54CD;&#x3002;alphaBgTx-5-&#x7F9F;&#x8272;&#x80FA; (3A) Rs&#xFF0C;&#x51C0;&#x5316;&#x6BEB;&#x514B;&#x6570;&#x91CF;&#x548C;&#x5728;&#x5927;&#x7EA6; 25%&#x7EAF;&#x5EA6;&#x7684;&#x6D17;&#x6DA4;&#x5242;&#x63D0;&#x53D6; alphaBgTx &#x624B;&#x6027;&#x4EB2;&#x548C;&#x67F1;&#x4E0A;&#x3002;&#x758F;&#x6C34; photolabel 3-&#x4E09;&#x6C1F;&#x7532;&#x57FA;-3-(m-[(125) i] iodophenyl) diazirine ([(125) i] &#x5DE5;&#x8D38;&#x7F72;&#xFF09; &#x88AB;&#x7528;&#x6765;&#x67E5;&#x660E;&#x6C28;&#x57FA;&#x9178;&#x5728;&#x8102;&#x86CB;&#x767D;&#x754C;&#x9762;&#x7684;&#x7EAF;&#x51C0;&#x548C;&#x8102;&#x8D28;&#x91CD;&#x7EC4; alphaBgTx-5 HT (3A) Rs&#x3002; [(125) i] &#x5DE5;&#x8D38;&#x7F72; photoincorporation &#x5230; alphaBgTx-5-&#x7F9F;&#x8272;&#x80FA; (3A) R &#x4E9A;&#x57FA;&#x6700;&#x521D;&#x88AB;&#x6620;&#x5C04;&#x5230;&#x4E9A;&#x57FA;&#x86CB;&#x767D;&#x6C34;&#x89E3;&#x7247;&#x6BB5;&#x7684; 8 kDa&#x3001; &#x542B; M4 &#x8DE8;&#x819C;&#x6BB5;&#x548C;&#x5927;&#x7EA6; 60%&#x7684;&#x7EB3;&#x5165;&#x6211; (125)&#x548C; 17 kDa&#xFF0C;&#x5305;&#x542B;&#x7684; M1 M3 &#x8DE8;&#x819C;&#x6BB5;&#x3002;M4 &#x6BB5;&#xFF0C;[(125) i] &#x5185;&#x5DE5;&#x8D38;&#x6807;&#x6709; Ser(451)&#xFF0C;&#x76F8;&#x5F53;&#x4E8E;&#x4E3A; [(125) I] &#x5DE5;&#x8D38;&#x6807;&#x6709;&#x6B8B;&#x7559; Thr(422) &#x9C7C;&#x96F7;&#x70DF;&#x78B1;&#x578B;&#x4E59;&#x9170;&#x80C6;&#x78B1;&#x53D7;&#x4F53; &#xFF08;nAChR&#xFF09; alpha1M4 &alpha;-&#x87BA;&#x65CB;&#x8102;&#x8D28;&#x66B4;&#x9732;&#x9762;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x63D0;&#x4F9B;&#x7684; 5-&#x7F9F;&#x8272;&#x80FA; (3A) &#x8868;&#x9762;&#x7684;&#x7B2C;&#x4E00;&#x5B9A;&#x4E49; R M4 &#x87BA;&#x65CB;&#xFF0C;&#x66B4;&#x9732;&#x4E8E;&#x8102;&#x8D28;&#x548C;&#x5EFA;&#x7ACB;&#x6B64;&#x8868;&#x9762;&#x662F;&#x7B49;&#x6548;&#x4E8E;&#x5728;&#x9C7C;&#x96F7; nAChR &#x8102;&#x8D28;&#x7684;&#x9762;&#x66B4;&#x9732;&#x3002;

&#x758F;&#x6C34;&#x6027;&#x5149;&#x654F;&#x7814;&#x7A76;&#x786E;&#x5B9A; 5 HT3A &#x53D7;&#x4F53;&#x7684;&#x8102;&#x86CB;&#x767D;&#x754C;&#x9762;&#x3002;

A HEK-293 cell line that stably expresses mouse 5-HT(3A)Rs containing a C-terminal extension that confers high-affinity binding of alpha-bungarotoxin (alphaBgTx) was established (alphaBgTx-5-HT(3A)Rs) and used to purify alphaBgTx-5-HT(3A)Rs in a lipid environment for use in structural studies using photoaffinity labeling. alphaBgTx-5-HT(3A)Rs were expressed robustly (60 pmol of [(3)H]BRL-43694 binding sites (approximately 3 microg of receptor) per milligram of protein) and displayed the same functional properties as wild-type receptors (serotonin EC(50) = 5.3 +/- 0.04 microM). While [(125)I]alphaBgTx bound to the alphaBgTx-5-HT(3A)Rs with high affinity (K(d) = 11 nM), application of nonradioactive alphaBgTx (up to 300 microM) had no effect on serotonin-induced current responses. alphaBgTx-5-HT(3A)Rs were purified on an alphaBgTx-derivatized affinity column from detergent extracts in milligram quantities and at approximately 25% purity. The hydrophobic photolabel 3-trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) was used to identify the amino acids at the lipid-protein interface of purified and lipid-reconstituted alphaBgTx-5-HT(3A)Rs. [(125)I]TID photoincorporation into the alphaBgTx-5-HT(3A)R subunit was initially mapped to subunit proteolytic fragments of 8 kDa, containing the M4 transmembrane segment and approximately 60% of incorporated (125)I, and 17 kDa, containing the M1-M3 transmembrane segments. Within the M4 segment, [(125)I]TID labeled Ser(451), equivalent to the [(125)I]TID-labeled residue Thr(422) at the lipid-exposed face of the Torpedo nicotinic acetylcholine receptor (nAChR) alpha1M4 alpha-helix. These results provide a first definition of the surface of the 5-HT(3A)R M4 helix that is exposed to lipid and establish that this surface is equivalent to the surface exposed to lipid in the Torpedo nAChR.

Hydrophobic photolabeling studies identify the lipid-protein interface of the 5-HT3A receptor.

Une ligne HEK-293 cellule qui exprime de fa&#xE7;on stable la souris 5-HT (3A) Rs contenant une extension C-terminale qui conf&#xE8;re &#xE0; haute affinit&#xE9; de liaison de l&#x26;#39;alpha-bungarotoxine (alphaBgTx) a &#xE9;t&#xE9; cr&#xE9;&#xE9; (alphaBgTx-5-HT (3A) Rs) et utilis&#xE9; pour purifier alphaBgTx-5-HT (3A) Rs dans un environnement lipidique pour une utilisation dans des &#xE9;tudes structurales &#xE0; l&#x26;#39;aide de marquage par photoaffinit&#xE9;. alphaBgTx-5-HT (3A) Rs ont &#xE9;t&#xE9; exprim&#xE9;es robuste (60 pmol de [(3) H] BRL-43694 sites de liaison (environ 3 microgrammes de r&#xE9;cepteur) par milligramme de prot&#xE9;ine) et affich&#xE9; les m&#xEA;mes propri&#xE9;t&#xE9;s fonctionnelles que de type sauvage r&#xE9;cepteurs (s&#xE9;rotonine CE (50) = 5,3 + / - 0,04 microM). Alors que [(125) I] alphaBgTx li&#xE9; &#xE0; la alphaBgTx-5-HT (3A) avec Rs haute affinit&#xE9; (K (d) = 11 nM), l&#x26;#39;application de non radioactif alphaBgTx (jusqu&#x26;#39;&#xE0; 300 microM) n&#x26;#39;a eu aucun effet sur la s&#xE9;rotonine induite par les r&#xE9;ponses actuelles. alphaBgTx-5-HT (3A) Rs ont &#xE9;t&#xE9; purifi&#xE9;s sur une colonne d&#x26;#39;affinit&#xE9; alphaBgTx-d&#xE9;riv&#xE9; &#xE0; partir d&#x26;#39;extraits de d&#xE9;tergent dans milligrammes et &#xE0; environ 25% de puret&#xE9;. Le hydrophobe photolabel 3-trifluorom&#xE9;thyl-3-(m-[(125) I] iodoph&#xE9;nyl) diazirine ([(125) I] TID) a &#xE9;t&#xE9; utilis&#xE9;e pour identifier les acides amin&#xE9;s &#xE0; l&#x26;#39;interface lipide-prot&#xE9;ine purifi&#xE9;e et de lipides-reconstitu&#xE9; alphaBgTx -5-HT (3A) Rs. [(125) I] TID photoincorporation dans le alphaBgTx-5-HT (3A) sous-unit&#xE9; a &#xE9;t&#xE9; initialement R mapp&#xE9; &#xE0; des fragments de sous-unit&#xE9;s prot&#xE9;olytiques de 8 kDa, contenant le segment transmembranaire M4 et environ 60% de incorpor&#xE9;e (125) I, et 17 kDa , contenant des segments transmembranaires M1-M3. L&#x26;#39;int&#xE9;rieur du segment M4, [(125) I] TID marqu&#xE9; Ser (451), &#xE9;quivalente &#xE0; la [(125) I] TID marqu&#xE9; r&#xE9;sidu Thr (422) &#xE0; la face expos&#xE9;e de lipides r&#xE9;cepteur de l&#x26;#39;ac&#xE9;tylcholine nicotinique torpille (nAChR) alpha1M4 h&#xE9;lice alpha. Les r&#xE9;sultats de fournir une premi&#xE8;re d&#xE9;finition de la surface de l&#x26;#39;h&#xE9;lice de 5-HT (3A) R M4 qui est expos&#xE9;e &#xE0; des lipides et &#xE9;tablir que cette surface est &#xE9;quivalente &#xE0; la surface expos&#xE9;e &#xE0; des lipides dans le nAChR torpille.

Des &#xE9;tudes photomarquage hydrophobes Identifier l&#x26;#39;interface lipide-prot&#xE9;ine du r&#xE9;cepteur 5-HT3A

Eine Zelllinie HEK-293, dass stabil dr&uuml;ckt 5-HT (3A) Rs Maus, enth&auml;lt eine Erweiterung der C-terminalen, hoher Affinit&auml;t Schwerg&auml;ngigkeit des Alpha-Bungarotoxin (AlphaBgTx) verleiht, gegr&uuml;ndet (alphaBgTx-5-HT(3A)Rs) und AlphaBgTx-5-HT (3A) Rs in einer Lipid-Umgebung f&uuml;r den Einsatz in strukturelle Studien mit Photoaffinity Kennzeichnung zu reinigen. AlphaBgTx-5-HT (3A) Rs waren robust ausgedr&uuml;ckt (60 Pmol von [(3) H] BRL-43694 Bindungsstellen (ca. 3 &micro;g des Rezeptors) pro Milligramm Protein) und die gleichen funktionellen Eigenschaften als Wildtyp-Rezeptoren (Serotonin EC(50) = 5.3 +/-0,04 MicroM). W&auml;hrend [(125) i] gebunden AlphaBgTx zu den AlphaBgTx-5-HT (3A) Rs mit hoher Affinit&auml;t (K(d) = 11 nM), Anwendung des eingeschmolzenen AlphaBgTx (bis zu 300 MicroM) hatte keinen Effekt auf die Serotonin-induzierte aktuelle Antworten. AlphaBgTx-5-HT (3A) Rs wurden auf einer AlphaBgTx-derivatized-Affinit&auml;tss&auml;ule Sp&uuml;lmittel Extrakte in Milligramm-Mengen und zu ca. 25 % Reinheit gereinigt. Die hydrophobe Photolabel 3-trifluormethyl - 3-(m-[(125) i] Iodophenyl) Diazirine ([(125) i] TID) wurde verwendet, um die Aminos&auml;uren an der Lipid-Protein-Schnittstelle von gereinigtem und Lipid-rekonstituiert AlphaBgTx-5-HT (3A) Rs. identifizieren [(125) i] TID Photoincorporation in der AlphaBgTx-5-HT (3A) R Untereinheit war zun&auml;chst Untereinheit proteolytische Fragmente von 8 kDa zugeordnet, mit der M4 Transmembran-Segment und etwa 60 % der aufgenommen (125) ich, und 17 kDa, mit die M1-M3-Transmembran-Segmenten. Im Segment M4, [(125) i] TID mit der Bezeichnung Ser(451), &auml;quivalent zu [(125) I] TID-beschrifteten R&uuml;ckstand Thr(422) auf das Lipid-gemachten Gesicht der Torpedo nikotinische Acetylcholin-Rezeptor (nAChR) alpha1M4 Alpha-Helix. Diese Ergebnisse liefern eine erste Definition der Oberfl&auml;che des 5-HT (3A) R M4-Helix, die Lipid ausgesetzt ist, und nachweisen, dass diese Fl&auml;che der Oberfl&auml;che ausgesetzt zu Lipid in der Torpedo-nAChR entspricht.

Hydrophobe Photolabeling Studien identifizieren die Lipid-Protein-Schnittstelle des Rezeptors 5-HT3A.

Fu stabilita una linea cellulare HEK-293 che stabilmente esprime il mouse Rs 5-HT (3A) contenente un&#39;estensione del C-terminale che conferisce ad alta affinit&agrave; legame di alfa-bungarotoxin (alphaBgTx) (alphaBgTx-5-HT(3A)Rs) e utilizzato per purificare Rs-alphaBgTx-5-HT (3A) in un ambiente lipidico per l&#39;utilizzo in studi strutturali utilizzando photoaffinity di etichettatura. Rs alphaBgTx-5-HT (3A) sono state espresse robustamente (60 pmol di siti di BRL 43694 leganti [3 H] (circa 3 microg del recettore) per milligrammo di proteine) e visualizzate le stesse propriet&agrave; funzionali come recettori di tipo selvatico (serotonina EC(50) = 5.3 + /-0,04 microM). Mentre [(125) i] alphaBgTx associata a alphaBgTx-5-HT (3A) Rs con alta affinit&agrave; (K(d) = 11 nM), applicazione di alphaBgTx non radioattivi (fino a 300 microM) ha avuto alcun effetto sulle risposte di corrente indotta da serotonina. Rs alphaBgTx-5-HT (3A) sono stati purificati in una colonna di affinit&agrave; derivatizzata alphaBgTx da detergente estratti in quantit&agrave; milligrammi e a circa 25% di purezza. Il photolabel idrofobici 3-trifluoromethyl - 3-(m-[(125) i] iodophenyl) diazirine ([(125) i] TID) &egrave; stato utilizzato per identificare gli aminoacidi all&#39;interfaccia lipido-proteina di purificato e ricostituito in lipidi alphaBgTx-5-HT (3A) Rs. [(125) i] TID photoincorporation in alphaBgTx-5-HT (3A) subunit&agrave; R inizialmente &egrave; stato mappato in frammenti proteolitici subunit&agrave; di 8 kDa, contenente la M4 segmento transmembrana e circa il 60% di incorporate I (125)e 17 kDa, contenente i segmenti transmembrana M1-M3. All&#39;interno del segmento M4, [(125) i] TID etichettati Ser(451), equivalente al [(125) I] TID-etichetta residuo Thr(422) alla faccia dei lipidi-esposti di siluro nicotinici acetilcolina recettore (nAChR) alpha1M4 alfa-elica. Questi risultati forniscono una prima definizione della superficie della 5-HT (3A) elica M4 R che &egrave; esposto a lipidi e stabilire che questa superficie &egrave; equivalente alla superficie esposta dei lipidi in nAChR il siluro.

Studi photolabeling idrofobici identificano l&#39;interfaccia di lipido-proteina del recettore 5-HT3A.

&#x5B89;&#x5B9A;&#x3057;&#x305F;&#x3B1;-&#x30D6;&#x30F3;&#x30AC;&#x30ED;&#x30C8;&#x30AD;&#x30B7;&#x30F3;&#xFF08;alphaBgTx&#xFF09;&#x306E;&#x9AD8;&#x89AA;&#x548C;&#x6027;&#x7D50;&#x5408;&#x3092;&#x4E0E;&#x3048;&#x308B;C&#x672B;&#x7AEF;&#x306E;&#x62E1;&#x5F35;&#x5B50;&#x3092;&#x542B;&#x3080;5-HT&#xFF08;3A&#xFF09;Rs&#x306F;&#xFF08;alphaBgTx-5-HT&#xFF08;3A&#xFF09;Rs&#xFF09;&#x304C;&#x8A2D;&#x7ACB;&#x3055;&#x308C;&#x305F;&#x30DE;&#x30A6;&#x30B9;&#x3092;&#x8868;&#x73FE;&#x3057;&#x3066;HEK-293&#x7D30;&#x80DE;&#x682A;&#x3068;&#x5149;&#x89AA;&#x548C;&#x6027;&#x6A19;&#x8B58;&#x3092;&#x7528;&#x3044;&#x305F;&#x69CB;&#x9020;&#x89E3;&#x6790;&#x3067;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x8102;&#x8CEA;&#x74B0;&#x5883;&#x3067;alphaBgTx-5-HT&#xFF08;3A&#xFF09;RS&#x3092;&#x6D44;&#x5316;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002; alphaBgTx-5-HT&#xFF08;3A&#xFF09;Rs&#x306F;&#x78BA;&#x5B9F;&#x306B;&#x767A;&#x73FE;&#x3059;&#x308B;&#xFF08;60 pmol&#x306E;[3 H]&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x30DF;&#x30EA;&#x30B0;&#x30E9;&#x30E0;&#x5F53;&#x305F;&#x308A;BRL-43694&#x7D50;&#x5408;&#x90E8;&#x4F4D;&#xFF08;&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x7D04;3&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#xFF09;&#xFF09;&#x3001;&#x304A;&#x3088;&#x3073;&#x91CE;&#x751F;&#x578B;&#x53D7;&#x5BB9;&#x4F53;&#x3068;&#x540C;&#x3058;&#x6A5F;&#x80FD;&#x306E;&#x30D7;&#x30ED;&#x30D1;&#x30C6;&#x30A3;&#x3092;&#x8868;&#x793A;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3057;&#x305F;&#xFF08;&#x30BB;&#x30ED;&#x30C8;&#x30CB;&#x30F3;EC&#xFF08;50&#xFF09;= 5.3 + /  -  0.04&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x30E2;&#x30EB;&#xFF09;&#x3002;&#x4E00;&#x65B9;&#x3001;[&#xFF08;125&#xFF09;I]&#x9AD8;&#x89AA;&#x548C;&#x6027;&#xFF08;K&#xFF08;D&#xFF09;= 11 nM&#xFF09;&#x3092;&#x6301;&#x3064;alphaBgTx-5-HT&#xFF08;3A&#xFF09;RS&#x306B;&#x30D0;&#x30A4;&#x30F3;&#x30C9;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;alphaBgTx&#x306F;&#x3001;&#x975E;&#x653E;&#x5C04;&#x6027;alphaBgTx&#xFF08;&#x6700;&#x5927;300&#x30DE;&#x30A4;&#x30AF;&#x30ED;&#x30E2;&#x30EB;&#xFF09;&#x306E;&#x30A2;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3067;&#x306F;&#x3001;&#x30BB;&#x30ED;&#x30C8;&#x30CB;&#x30F3;&#x8A98;&#x767A;&#x6027;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x53CA;&#x307C;&#x3055;&#x306A;&#x304B;&#x3063;&#x305F;&#x73FE;&#x5728;&#x306E;&#x5FDC;&#x7B54;&#x3002; alphaBgTx-5-HT&#xFF08;3A&#xFF09;Rs&#x306F;&#x30DF;&#x30EA;&#x30B0;&#x30E9;&#x30E0;&#x91CF;&#x306E;&#x7D04;25&#xFF05;&#x306E;&#x7D14;&#x5EA6;&#x3067;&#x6D17;&#x5264;&#x62BD;&#x51FA;&#x7269;&#x304B;&#x3089;alphaBgTx&#x8A98;&#x5C0E;&#x4F53;&#x5316;&#x30A2;&#x30D5;&#x30A3;&#x30CB;&#x30C6;&#x30A3;&#x30FC;&#x30AB;&#x30E9;&#x30E0;&#x3067;&#x7CBE;&#x88FD;&#x3057;&#x305F;&#x3002;&#x758E;&#x6C34;&#x6027;photolabel 3  - &#x30C8;&#x30EA;&#x30D5;&#x30EB;&#x30AA;&#x30ED;&#x30E1;&#x30C1;&#x30EB;-3  - &#xFF08;M-[&#xFF08;125&#xFF09;I]&#x30E8;&#x30FC;&#x30C9;&#x30D5;&#x30A7;&#x30CB;&#x30EB;&#xFF09;&#x30B8;&#x30A2;&#x30B8;&#x30EA;&#x30F3;&#xFF08;[&#xFF08;125&#xFF09;I] TID&#xFF09;&#x306F;&#x3001;&#x7CBE;&#x88FD;&#x3057;&#x3001;&#x8102;&#x8CEA;&#x518D;&#x69CB;&#x6210;alphaBgTx&#x306E;&#x8102;&#x8CEA;&#x86CB;&#x767D;&#x8CEA;&#x754C;&#x9762;&#x3067;&#x306E;&#x30A2;&#x30DF;&#x30CE;&#x9178;&#x3092;&#x540C;&#x5B9A;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x305F;-5-HT&#xFF08;3A&#xFF09;&#x306B;&#x306F;&#x30B5;&#x30A4;&#x30F3;&#x30A4;&#x30F3;&#x3057;&#x3066;&#x304F;&#x3060;&#x3055;&#x3044;&#x3002; [&#xFF08;125&#xFF09;I] TID&#x306E;photoincorporation alphaBgTx-5-HT&#xFF08;3A&#xFF09;R&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x304C;&#x6700;&#x521D;&#x306B;M4&#x306E;&#x819C;&#x8CAB;&#x901A;&#x30BB;&#x30B0;&#x30E1;&#x30F3;&#x30C8;&#x3068;&#x6CD5;&#x4EBA;&#x306E;&#x7D04;60&#xFF05;&#xFF08;125&#xFF09;&#x79C1;&#x306F;&#x3001;17 kDa&#x306E;&#x3092;&#x542B;&#x3080;&#x3001;8 kDa&#x306E;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x5206;&#x89E3;&#x30D5;&#x30E9;&#x30B0;&#x30E1;&#x30F3;&#x30C8;&#x306B;&#x30DE;&#x30C3;&#x30D7;&#x3055;&#x308C;&#x305F;&#x306B;&#x3001;M1-M3&#x8CAB;&#x901A;&#x30BB;&#x30B0;&#x30E1;&#x30F3;&#x30C8;&#x3092;&#x542B;&#x3080;&#x3002; M4&#x30BB;&#x30B0;&#x30E1;&#x30F3;&#x30C8;&#x5185;&#x3067;&#x3001;[&#xFF08;125&#xFF09;I]&#x306B;&#x76F8;&#x5F53;&#x3059;&#x308B;TID&#x30E9;&#x30D9;&#x30EB;SER&#xFF08;451&#xFF09;&#x3001;[&#xFF08;125&#xFF09;I]&#x9B5A;&#x96F7;&#x30CB;&#x30B3;&#x30C1;&#x30F3;&#x6027;&#x30A2;&#x30BB;&#x30C1;&#x30EB;&#x30B3;&#x30EA;&#x30F3;&#x53D7;&#x5BB9;&#x4F53;&#xFF08;nAChR&#xFF09;&#x306E;&#x8102;&#x8CEA;&#x9732;&#x51FA;&#x9762;&#x306B;&#x304A;&#x3051;&#x308B;&#x6B8B;&#x6E23;&#x306E;Thr&#xFF08;422&#xFF09;TID-&#x6A19;&#x8B58;alpha1M4&#x3B1;-&#x30D8;&#x30EA;&#x30C3;&#x30AF;&#x30B9;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x306F;&#x3001;&#x8102;&#x8CEA;&#x306B;&#x3055;&#x3089;&#x3055;&#x308C;&#x308B;&#x3068;&#x3001;&#x3053;&#x306E;&#x8868;&#x9762;&#x306F;&#x9B5A;&#x96F7;nAChR&#x306E;&#x4E2D;&#x306E;&#x8102;&#x8CEA;&#x306B;&#x3055;&#x3089;&#x3055;&#x308C;&#x308B;&#x9762;&#x3068;&#x7B49;&#x4FA1;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x304C;&#x78BA;&#x7ACB;&#x3055;&#x308C;&#x305F;5-HT&#xFF08;3A&#xFF09;R M4&#x3078;&#x30EA;&#x30C3;&#x30AF;&#x30B9;&#x306E;&#x8868;&#x9762;&#x306E;&#x6700;&#x521D;&#x306E;&#x5B9A;&#x7FA9;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x758E;&#x6C34;&#x6027;&#x306E;&#x5149;&#x6A19;&#x8B58;&#x7814;&#x7A76;&#x306F;&#x3001;5 HT3A&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x8102;&#x8CEA; - &#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30D5;&#x30A7;&#x30A4;&#x30B9;&#x3092;&#x8B58;&#x5225;

&#xC548;&#xC815;&#xC801;&#xC73C;&#xB85C; &#xD45C;&#xD604; &#xD558; &#xACE0; &#xC54C;&#xD30C;-bungarotoxin (alphaBgTx)&#xC758; &#xB192;&#xC740; &#xCE5C; &#xD654;&#xB825; &#xBC14;&#xC778;&#xB529; &#xC218; &#xC5EC; C &#xD130;&#xBBF8;&#xB110; &#xD655;&#xC7A5;&#xC744; &#xD3EC;&#xD568; &#xD558;&#xB294; 5-HT (3A) Rs &#xB9C8;&#xC6B0;&#xC2A4; HEK 293 &#xC138;&#xD3EC; &#xB77C;&#xC778;&#xC744; &#xC124;&#xB9BD; (alphaBgTx-5-HT(3A)Rs) photoaffinity &#xB77C;&#xBCA8;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xAD6C;&#xC870; &#xC5F0;&#xAD6C;&#xC5D0; &#xC0AC;&#xC6A9; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC9C0;&#xC9C8; &#xD658;&#xACBD;&#xC5D0;&#xC11C; alphaBgTx-5-HT (3A) Rs&#xB97C; &#xC815;&#xD654; &#xD558;&#xB294; &#xB370; &#xC0AC;&#xC6A9; &#xD569;&#xB2C8;&#xB2E4;. alphaBgTx-5-HT (3A) Rs &#xACAC;&#xACE0; &#xD558; &#xAC8C; &#xD45C;&#xD604; &#xD588;&#xB2E4; (60 &#xBC00;&#xB9AC; &#xADF8;&#xB7A8;&#xC758; &#xB2E8;&#xBC31;&#xC9C8; &#xB2F9; [(3) H] BRL 43694 &#xBC14;&#xC778;&#xB529; &#xC0AC;&#xC774;&#xD2B8; (&#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xC57D; 3 microg) pmol) &#xC57C;&#xC0DD; &#xD615; &#xC218;&#xC6A9; &#xCCB4;&#xC640; &#xB3D9;&#xC77C;&#xD55C; &#xAE30;&#xB2A5; &#xC18D;&#xC131;&#xC744; &#xD45C;&#xC2DC; &#xD558; &#xACE0; (&#xC138;&#xB85C;&#xD1A0;&#xB2CC; EC(50) = 0.04 microM + 5.3). [&#xC804; (125)] &#xB3D9;&#xC548; Alphabgtx&#xC5D0; &#xBC14;&#xC778;&#xB529;&#xB41C; alphaBgTx 5 HT (3A) Rs &#xB192;&#xC740; &#xC120;&#xD638;&#xB3C4; (K(d) = 11 nM), nonradioactive alphaBgTx (&#xCD5C;&#xB300; 300 microM)&#xC758; &#xC801;&#xC6A9; &#xD588;&#xB2E4; &#xD604;&#xC7AC; &#xC751;&#xB2F5; &#xC138;&#xB85C;&#xD1A0;&#xB2CC; &#xC720;&#xBC1C;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xC8FC;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;. alphaBgTx-5-HT (3A) Rs &#xBC00;&#xB9AC; &#xADF8;&#xB7A8; &#xC218;&#xB7C9; &#xBC0F; &#xC57D; 25% &#xC21C;&#xB3C4;&#xC5D0; &#xC138;&#xC81C; &#xCD94;&#xCD9C; &#xBB3C;&#xC5D0;&#xC11C; alphaBgTx derivatized &#xC120;&#xD638;&#xB3C4; &#xC5F4; &#xC21C;&#xD654; &#xD588;&#xB2E4;. &#xC18C;&#xC218; photolabel 3-trifluoromethyl-3-(m-[I (125)] iodophenyl) diazirine ([I (125)] TID)&#xC758; &#xC21C;&#xD654; &#xBC0F; &#xC9C0;&#xC9C8; &#xC7AC;&#xAD6C;&#xC131; alphaBgTx-5-HT (3A) Rs. &#xC9C0;&#xC9C8; &#xB2E8;&#xBC31;&#xC9C8; &#xC778;&#xD130;&#xD398;&#xC774;&#xC2A4;&#xC5D0;&#xC11C; &#xC544;&#xBBF8;&#xB178;&#xC0B0;&#xC744; &#xC2DD;&#xBCC4; &#xD558;&#xB294; &#xB370; &#xC0AC;&#xC6A9;&#xD55C; [I (125)] alphaBgTx 5 HT (3A)&#xC5D0; TID photoincorporation R &#xC18C; &#xB2E8;&#xC704; 8 Kda&#xC758; &#xC18C; &#xB2E8;&#xC704; &#xB2E8;&#xBC31;&#xC9C8; &#xC870;&#xAC01;&#xC5D0; &#xB9E4;&#xD551;&#xB41C; &#xCC98;&#xC74C; &#xD3EC;&#xD568; &#xB41C; m 4 &#xB9C9; &#xD6A1;&#xB2E8; &#xC138;&#xADF8;&#xBA3C;&#xD2B8; &#xBC0F;&#xC758; &#xC57D; 60% (125) &#xB098; &#xD1B5;&#xD569;,&#xADF8;&#xB9AC;&#xACE0; M1 M3 &#xB9C9; &#xD6A1;&#xB2E8; &#xC138;&#xADF8;&#xBA3C;&#xD2B8;&#xB97C; &#xD3EC;&#xD568; &#xD558;&#xB294; 17 kDa. [&#xC804; (125)] M4 &#xC138;&#xADF8;&#xBA3C;&#xD2B8; &#xB0B4;&#xC5D0;&#xC11C; TID &#xBD84;&#xB958; Ser(451), [(125) I]&#xC5D0; &#xD574;&#xB2F9; TID &#xD45C;&#xC2DC; &#xB41C; Thr(422) &#xC794;&#xB958;&#xBB3C;&#xC758; &#xC5B4; &#xB8B0; nicotinic &#xC544; &#xC138; &#xD2F8; &#xCF5C;&#xB9B0; &#xC218;&#xC6A9; &#xCCB4; (nAChR) alpha1M4 &#xC54C;&#xD30C; &#xB098;&#xC120; &#xC9C0;&#xC9C8; &#xB178;&#xCD9C; &#xC5BC;&#xAD74;. 5-HT (3A)&#xC758; &#xCCAB; &#xBC88;&#xC9F8; &#xC815;&#xC758; &#xC81C;&#xACF5; &#xD558;&#xB294; &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC; &#xC9C0;&#xC9C8;&#xC5D0; &#xB178;&#xCD9C; &#xB418; &#xACE0;&#xC774; &#xD45C;&#xBA74;&#xC5D0;&#xC5D0; &#xC5B4; &#xB8B0; nAChR &#xC9C0;&#xC9C8; &#xB178;&#xCD9C; &#xD45C;&#xBA74;&#xC5D0; &#xD574;&#xB2F9; &#xD558;&#xB294; R m 4 &#xB098;&#xC120;.

&#xC18C;&#xC218; &#xC131; photolabeling &#xC5F0;&#xAD6C; 5-HT3A &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xC9C0;&#xC9C8; &#xB2E8;&#xBC31;&#xC9C8; &#xC778;&#xD130;&#xD398;&#xC774;&#xC2A4;&#xB97C; &#xC2DD;&#xBCC4; &#xD569;&#xB2C8;&#xB2E4;.

Un HEK-293 l&#xED;nea celular que expresa establemente rat&#xF3;n de 5-HT (3A) R contiene una extensi&#xF3;n C-terminal que confiere alta afinidad de uni&#xF3;n del alfa-bungarotoxina (alphaBgTx) fue establecida (alphaBgTx-5-HT (3A) Rs) y utilizado para purificar alphaBgTx-5-HT (3A) Rs en un entorno de l&#xED;pidos para su uso en estudios estructurales utilizando etiquetado fotoafinidad. alphaBgTx-5-HT (3A) R se expresaron robustamente (60 pmol de [(3) H]-BRL 43694 sitios de uni&#xF3;n (aproximadamente 3 microgramos de receptor) por miligramo de prote&#xED;na) y se muestran las mismas propiedades funcionales como los receptores de tipo salvaje (serotonina CE (50) = 5,3 + / - 0,04 microM). Mientras [(125) I] alphaBgTx unido al alphaBgTx-5-HT (3A) R con alta afinidad (K (D) = 11 nM), la aplicaci&#xF3;n de no radiactivo alphaBgTx (hasta 300 microM) no tuvo ning&#xFA;n efecto sobre la serotonina inducida respuestas actuales. alphaBgTx-5-HT (3A) R se purificaron en una columna de afinidad alphaBgTx derivatizado a partir de extractos de detergente en cantidades de miligramos y en aproximadamente un 25% de pureza. El hidr&#xF3;fobo photolabel 3-trifluorometil-3-(m-[(125) I] yodofenil) diazirine ([(125) I] TID) se utiliz&#xF3; para identificar los amino&#xE1;cidos en la interfase l&#xED;pido-prote&#xED;na purificada de l&#xED;pidos y reconstituida-alphaBgTx -5-HT (3A) Rs. [(125) I] photoincorporation TID en la alphaBgTx-5-HT (3A) subunidad R se asigna inicialmente a fragmentos proteol&#xED;ticos de subunidades de 8 kDa, que contiene el segmento transmembrana M4 y aproximadamente 60% de Incorporated (125) I, y 17 kDa , que contiene los segmentos de transmembrana M1-M3. Dentro del segmento M4, [(125) I] TID etiquetados Ser (451), equivalente al [(125) I] TID-etiquetados residuo Thr (422) en la cara expuesta de l&#xED;pido-receptor de acetilcolina Torpedo nicot&#xED;nico (nAChR) alpha1M4 alfa-h&#xE9;lice. Estos resultados proporcionan una primera definici&#xF3;n de la superficie de la 5-HT h&#xE9;lice (3A) R M4 que se expone a l&#xED;pidos y establecer que esta superficie es equivalente a la superficie expuesta a los l&#xED;pidos en la nAChR torpedo.

Estudios hidrof&#xF3;bicos Photolabeling identificar la interfaz de l&#xED;pido-prote&#xED;na del receptor de 5-HT3A

Mycobacterium tuberculosis survives in latently infected individuals, likely in a nonreplicating or dormancy-like state. The M. tuberculosis DosR regulon is a genetic program induced by conditions that inhibit aerobic respiration and prevent bacillus replication. In this study, we used a mutant incapable of DosR regulon induction to investigate the contribution of this regulon to bacterial metabolism during anaerobic dormancy. Our results confirm that the DosR regulon is essential for M. tuberculosis survival during anaerobic dormancy and demonstrate that it is required for metabolic processes that occur upon entry into and throughout the dormant state. Specifically, we showed that regulon mechanisms shift metabolism away from aerobic respiration in the face of dwindling oxygen availability and are required for maintaining energy levels and redox balance as the culture becomes anaerobic. We also demonstrated that the DosR regulon is crucial for rapid resumption of growth once M. tuberculosis exits an anaerobic or nitric oxide-induced nonrespiring state. In summary, the DosR regulon encodes novel metabolic mechanisms essential for M. tuberculosis to survive in the absence of respiration and to successfully transition rapidly between respiring and nonrespiring conditions without loss of viability.

The Mycobacterium tuberculosis DosR regulon assists in metabolic homeostasis and enables rapid recovery from nonrespiring dormancy.

Fluorescent proteins and molecules are now widely used to tag and visualize proteins resulting in an improved understanding of protein trafficking, localization, and function. In addition, fluorescent tags have also been used to inactivate protein function in a spatially and temporally-defined manner, using a technique known as fluorophore-assisted light inactivation (FALI) or chromophore-assisted light inactivation (CALI). In this study we tagged the serotoninâ‚ƒ A subunit with the Î±-bungarotoxin binding sequence (BBS) and subsequently labeled 5-HTâ‚ƒA/BBS receptors with fluorescently conjugated Î±-bungarotoxin in live cells. We show that 5-HTâ‚ƒA/BBS receptors are constitutively internalized in the absence of an agonist and internalization as well as receptor function are inhibited by fluorescence. The fluorescence-induced disruption of function and internalization was reduced with oxygen radical scavengers suggesting the involvement of reactive oxygen species, implicating the FALI process. Furthermore, these data suggest that intense illumination during live-cell microscopy may result in inadvertent FALI and inhibition of protein trafficking.

Fluorophore assisted light inactivation (FALI) of recombinant 5-HTâ‚ƒA receptor constitutive internalization and function.

&#x8367;&#x5149;&#x86CB;&#x767D;&#x548C;&#x5206;&#x5B50;&#x73B0;&#x5728;&#x5E7F;&#x6CDB;&#x7528;&#x4E8E;&#x6807;&#x8BB0;&#x548C;&#x53EF;&#x89C6;&#x5316;&#x86CB;&#x767D;&#x8D28;&#x5BFC;&#x81F4;&#x86CB;&#x767D;&#x8D28;&#x8D29;&#x8FD0;&#x3001; &#x672C;&#x5730;&#x5316;&#x548C;&#x529F;&#x80FD;&#x66F4;&#x597D;&#x5730;&#x4E86;&#x89E3;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x8367;&#x5149;&#x6807;&#x8BB0;&#x4E5F;&#x5DF2;&#x7528;&#x4E8E;&#x706D;&#x6D3B;&#x86CB;&#x767D;&#x529F;&#x80FD;&#x7684;&#x7A7A;&#x95F4;&#x548C;&#x6682;&#x65F6;&#x5B9A;&#x4E49;&#x7684;&#x65B9;&#x5F0F;&#xFF0C;&#x4F7F;&#x7528;&#x4E00;&#x79CD;&#x53EB;&#x505A;&#x8367;&#x5149;&#x8F85;&#x52A9;&#x5149;&#x706D;&#x6D3B; (&#x53D1;&#x529B;) &#x6216;&#x53D1;&#x8272;&#x56E2;&#x534F;&#x52A9;&#x5149;&#x706D;&#x6D3B; (&#x5361;&#x5229;)&#x3002;&#x5728;&#x8FD9;&#x9879;&#x7814;&#x7A76;&#x4E2D;&#x6211;&#x4EEC;&#x6807;&#x8BB0; serotonin&#x2083; A &#x4E9A;&#x57FA;&#x4E0E; &alpha;-&#x91D1;&#x73AF;&#x86C7;&#x6BD2;&#x7D20;&#x7ED1;&#x5B9A;&#x5E8F;&#x5217; (BBS) &#x548C;&#x540E;&#x6765;&#x6807;&#x8BB0; 5-HT&#x2083;A/BBS &#x4E0E;&#x9057;&#x4F20;&#x7269;&#x8D28;&#x5171;&#x8F6D; &alpha; &#x91D1;&#x73AF;&#x86C7;&#x6BD2;&#x7D20;&#x5728;&#x6D3B;&#x7EC6;&#x80DE;&#x4E2D;&#x7684;&#x53D7;&#x4F53;&#x3002;&#x6211;&#x4EEC;&#x5C55;&#x793A; 5-HT&#x2083;A/BBS &#x53D7;&#x4F53;&#x88AB;&#x4E00;&#x6FC0;&#x52A8;&#x5242;&#x6CA1;&#x6709;&#x5185;&#x5728;&#x5316;&#x548C;&#x56FD;&#x9645;&#x5316;&#xFF0C;&#x4EE5;&#x53CA;&#x53D7;&#x4F53;&#x529F;&#x80FD;&#x5747;&#x7531;&#x8367;&#x5149;&#x6709;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x3002;&#x4E0E;&#x6C27;&#x81EA;&#x7531;&#x57FA;&#x62FE;&#x8352;&#x6697;&#x793A;&#x7684;&#x6D3B;&#x6027;&#x6C27;&#xFF0C;&#x53C2;&#x4E0E;&#x6709;&#x7275;&#x8FDE;&#x7684;&#x53D1;&#x529B;&#x8FC7;&#x7A0B;&#x51CF;&#x5C11;&#x4E86;&#x8BF1;&#x5BFC;&#x8367;&#x5149;&#x7684;&#x4E2D;&#x65AD;&#x529F;&#x80FD;&#x548C;&#x5185;&#x5728;&#x5316;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x8FD9;&#x4E9B;&#x6570;&#x636E;&#x8868;&#x660E;&#x6FC0;&#x70C8;&#x7167;&#x5EA6;&#x5728;&#x6D3B;&#x7EC6;&#x80DE;&#x663E;&#x5FAE;&#x955C;&#x53EF;&#x80FD;&#x5728;&#x65E0;&#x610F;&#x4E2D;&#x53D1;&#x529B;&#x548C;&#x6291;&#x5236;&#x86CB;&#x767D;&#x8D29;&#x5356;&#x4EBA;&#x53E3;&#x3002;

&#x8367;&#x5149;&#x8F85;&#x52A9;&#x5149;&#x706D;&#x6D3B; (&#x53D1;&#x529B;) &#x91CD;&#x7EC4; 5 HT&#x2083;A &#x53D7;&#x4F53;&#x672C;&#x6784;&#x5185;&#x90E8;&#x5316;&#x4E0E;&#x529F;&#x80FD;&#x3002;

Les prot&#xE9;ines fluorescentes et les mol&#xE9;cules sont maintenant largement utilis&#xE9;e pour baliser et de visualiser des prot&#xE9;ines r&#xE9;sultant d&#x26;#39;une meilleure compr&#xE9;hension de la traite des prot&#xE9;ines, la localisation et la fonction. En outre, les marqueurs fluorescents ont &#xE9;galement &#xE9;t&#xE9; utilis&#xE9;s pour inactiver la fonction des prot&#xE9;ines d&#x26;#39;une mani&#xE8;re spatialement et temporellement d&#xE9;fini, en utilisant une technique connue sous le nom fluorophore assist&#xE9;e par l&#x26;#39;inactivation de lumi&#xE8;re (Fali) ou l&#x26;#39;inactivation de lumi&#xE8;re chromophore assist&#xE9;e (CALI). Dans cette &#xE9;tude nous avons marqu&#xE9; la s&#xE9;rotonine &#x2083; a sous-unit&#xE9; avec la s&#xE9;quence &#x3B1;-bungarotoxine contraignant (BBS) et par la suite marqu&#xE9; 5-HT &#x2083; r&#xE9;cepteurs A / BBS avec fluorescence conjugu&#xE9; &#x3B1;-bungarotoxine dans les cellules vivantes. On montre que le 5-HT &#x2083; r&#xE9;cepteurs A BBS / sont constitutivement internalis&#xE9; en l&#x26;#39;absence d&#x26;#39;un agoniste et l&#x26;#39;internalisation ainsi que la fonction du r&#xE9;cepteur sont inhib&#xE9;s par fluorescence. La perturbation de fluorescence induite par la fonction et de l&#x26;#39;internalisation a &#xE9;t&#xE9; r&#xE9;duit avec des pi&#xE8;ges &#xE0; oxyg&#xE8;ne radicaux sugg&#xE9;rant l&#x26;#39;implication des esp&#xE8;ces r&#xE9;actives de l&#x26;#39;oxyg&#xE8;ne, impliquant le processus Fali. En outre, ces donn&#xE9;es sugg&#xE8;rent que l&#x26;#39;&#xE9;clairage intense pendant la microscopie des cellules vivantes peut entra&#xEE;ner par inadvertance Fali et l&#x26;#39;inhibition de la traite des prot&#xE9;ines.

Fluorophore assist&#xE9;e inactivation Lumi&#xE8;re (Fali) du recombinant 5-HT &#x2083; A l&#x26;#39;internalisation du r&#xE9;cepteur constitutif et de la fonction

Fluoreszierende Proteine und Molek&uuml;le werden nun allgemein zum-Tag und Proteine, was zu einem besseren Verst&auml;ndnis von Protein Menschenhandel, Lokalisierung und Funktion zu visualisieren. Dar&uuml;ber hinaus fluoreszierende Markierungen auch wurden verwendet, um inaktive Proteinfunktion in einer r&auml;umlich und zeitlich definierte Art und Weise, mit einer Technik bekannt als Fluorophor-assistierte Licht Inaktivierung (FALI) oder Chromophor-assistierte Licht Inaktivierung (CALI). In dieser Studie haben wir markiert die Serotonin&#x2083; A-Untereinheit mit der &alpha;-Bungarotoxin Bindung Sequenz (BBS) und anschlie&szlig;end mit der Bezeichnung 5-HT&#x2083;A/BBS-Rezeptoren mit Leuchtstoff konjugierte &alpha;-Bungarotoxin in lebenden Zellen. Wir zeigen, dass 5-HT&#x2083;A/BBS-Rezeptoren konstitutiv in Ermangelung ein Agonist verinnerlicht sind und Internalisierung sowie Rezeptor-Funktion durch Fluoreszenz gehemmt werden. Die Fluoreszenz-induzierte St&ouml;rung der Funktion und Internalisierung reduzierte sich mit Sauerstoff Radikale Aasfresser vorschlagen Einbeziehung der reaktive Sauerstoffspezies Nicht&uuml;bernahme des FALI-Prozess. Diese Daten zufolge dar&uuml;ber hinaus intensive Beleuchtung im Leben-Zelle Mikroskopie unbeabsichtigte FALI und Hemmung von Protein Handel f&uuml;hren kann.

Fluorophor unterst&uuml;tzt Licht Inaktivierung (FALI) von rekombinanten 5-HT&#x2083;A Rezeptor konstitutive Internalisierung und Funktion.

Molecole e proteine fluorescenti sono ora ampiamente usati di tag e di visualizzare le proteine risultanti in una migliore comprensione della proteina traffico, localizzazione e funzione. Inoltre, etichette fluorescenti sono stati utilizzati anche per inattivare la funzione della proteina in un spazialmente e temporalmente definiti modo, usando una tecnica conosciuta come inattivazione luce fluorophore-assistita (FALI) o inattivazione luce cromoforo-assistita (CALI). In questo studio abbiamo messo la subunit&agrave; serotonin&#x2083; A con la sequenza di legame &alpha;-bungarotoxin (BBS) e successivamente etichettati i recettori 5-HT&#x2083;A/BBS con fluorescente coniugati &alpha;-bungarotoxin in cellule vive. Mostriamo che i recettori 5-HT&#x2083;A/BBS sono costitutivamente interiorizzati in assenza di un agonista e interiorizzazione, come pure la funzione del recettore &egrave; inibiti dalla fluorescenza. La fluorescenza indotta interruzione di funzione e di interiorizzazione &egrave; stata ridotta con ossigeno radicale spazzini suggerendo il coinvolgimento di specie reattive dell&#39;ossigeno, implicando il processo FALI. Inoltre, questi dati suggeriscono che possa provocare involontaria FALI e inibizione della proteina tratta intensa illuminazione durante il live-cella microscopia.

Fluoroforo assistita luce inattivazione (FALI) di interiorizzazione costitutiva 5-HT&#x2083;A ricombinante del recettore e funzione.

&#x86CD;&#x5149;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3068;&#x5206;&#x5B50;&#x306F;&#x3001;&#x73FE;&#x5728;&#x5E83;&#x304F;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x8F38;&#x9001;&#x3001;&#x5C40;&#x5728;&#x5316;&#x3001;&#x304A;&#x3088;&#x3073;&#x6A5F;&#x80FD;&#x306E;&#x7406;&#x89E3;&#x306E;&#x5411;&#x4E0A;&#x3092;&#x3082;&#x305F;&#x3089;&#x3059;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x3092;&#x30BF;&#x30B0;&#x4ED8;&#x3051;&#x3057;&#x8996;&#x899A;&#x5316;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x86CD;&#x5149;&#x30BF;&#x30B0;&#x306F;&#x3001;&#x30D5;&#x30EB;&#x30AA;&#x30ED;&#x30D5;&#x30A9;&#x30A2;&#x56E3;&#x88DC;&#x52A9;&#x5149;&#x4E0D;&#x6D3B;&#x6027;&#x5316;&#xFF08;FALI&#xFF09;&#x307E;&#x305F;&#x306F;&#x767A;&#x8272;&#x56E3;&#x88DC;&#x52A9;&#x5149;&#x4E0D;&#x6D3B;&#x5316;&#xFF08;CALI&#xFF09;&#x3068;&#x3057;&#x3066;&#x77E5;&#x3089;&#x308C;&#x308B;&#x6280;&#x8853;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x7A7A;&#x9593;&#x7684;&#x304A;&#x3088;&#x3073;&#x6642;&#x9593;&#x7684;&#x306B;&#x5B9A;&#x7FA9;&#x3055;&#x308C;&#x305F;&#x65B9;&#x6CD5;&#x3067;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x6A5F;&#x80FD;&#x3092;&#x4E0D;&#x6D3B;&#x6027;&#x5316;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;&#x3B1;-&#x30D6;&#x30F3;&#x30AC;&#x30ED;&#x30C8;&#x30AD;&#x30B7;&#x30F3;&#x7D50;&#x5408;&#x914D;&#x5217;&#xFF08;BBS&#xFF09;&#x3067;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x3092;&#x2083;&#x30BB;&#x30ED;&#x30C8;&#x30CB;&#x30F3;&#x306B;&#x30BF;&#x30B0;&#x3092;&#x4ED8;&#x3051;&#x3001;&#x305D;&#x306E;&#x5F8C;&#x3001;&#x751F;&#x304D;&#x305F;&#x7D30;&#x80DE;&#x4E2D;&#x306E;&#x3B1;-&#x30D6;&#x30F3;&#x30AC;&#x30ED;&#x30C8;&#x30AD;&#x30B7;&#x30F3;&#x86CD;&#x5149;&#x30B3;&#x30F3;&#x30B8;&#x30E5;&#x30B2;&#x30FC;&#x30C8;&#x3068;5-HT&#x2083;/ BBS&#x53D7;&#x5BB9;&#x4F53;&#x3092;&#x6A19;&#x8B58;&#x3057;&#x305F;&#x3002;&#x6211;&#x3005;&#x306F;&#x86CD;&#x5149;&#x306B;&#x3088;&#x3063;&#x3066;&#x963B;&#x5BB3;&#x3055;&#x308C;&#x308B;&#x2083;/ BBS&#x53D7;&#x5BB9;&#x4F53;&#x304C;&#x6052;&#x5E38;&#x7684;&#x30A2;&#x30B4;&#x30CB;&#x30B9;&#x30C8;&#x304A;&#x3088;&#x3073;&#x5185;&#x5728;&#x5316;&#x306E;&#x6709;&#x7121;&#x3060;&#x3051;&#x3067;&#x306A;&#x304F;&#x3001;&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x6A5F;&#x80FD;&#x306B;&#x5185;&#x5728;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;5-HT&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x6A5F;&#x80FD;&#x3068;&#x5185;&#x5728;&#x306E;&#x86CD;&#x5149;&#x8A98;&#x8D77;&#x7834;&#x58CA;&#x306F;FALI&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x304C;&#x95A2;&#x4E0E;&#x3057;&#x3001;&#x6D3B;&#x6027;&#x9178;&#x7D20;&#x7A2E;&#x306E;&#x95A2;&#x4E0E;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x308B;&#x9178;&#x7D20;&#x30E9;&#x30B8;&#x30AB;&#x30EB;&#x30B9;&#x30AB;&#x30D9;&#x30F3;&#x30B8;&#x30E3;&#x30FC;&#x3067;&#x6E1B;&#x5C11;&#x3057;&#x305F;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x30C7;&#x30FC;&#x30BF;&#x306F;&#x3001;&#x751F;&#x7D30;&#x80DE;&#x9855;&#x5FAE;&#x93E1;&#x691C;&#x67FB;&#x6642;&#x306E;&#x5F37;&#x70C8;&#x306A;&#x7167;&#x660E;&#x304C;&#x4E0D;&#x6CE8;&#x610F;FALI&#x3068;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x8F38;&#x9001;&#x306E;&#x963B;&#x5BB3;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x305F;&#x3002;

&#x7D44;&#x63DB;&#x3048;5-HT&#x53D7;&#x5BB9;&#x4F53;&#x2083;&#x6052;&#x5E38;&#x7684;&#x5185;&#x5728;&#x5316;&#x3068;&#x6A5F;&#x80FD;&#x306E;&#x30D5;&#x30EB;&#x30AA;&#x30ED;&#x30D5;&#x30A9;&#x30A2;&#x88DC;&#x52A9;&#x5149;&#x4E0D;&#x6D3B;&#x5316;&#xFF08;FALI&#xFF09;

&#xD615;&#xAD11; &#xB2E8;&#xBC31;&#xC9C8; &#xBC0F; &#xBD84;&#xC790;&#xB294; &#xC9C0;&#xAE08; &#xB110;&#xB9AC; &#xC0AC;&#xC6A9; &#xD0DC;&#xADF8; &#xBC0F; &#xB2E8;&#xBC31;&#xC9C8; &#xB2E8;&#xBC31;&#xC9C8; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;, &#xC9C0;&#xC5ED;&#xD654;, &#xBC0F; &#xAE30;&#xB2A5; &#xD5A5;&#xC0C1; &#xB41C; &#xC774;&#xD574;&#xC758; &#xACB0;&#xACFC; &#xC2DC;&#xAC01;&#xD654;. &#xB610;&#xD55C;, &#xD615;&#xAD11; &#xD0DC;&#xADF8; &#xB610;&#xD55C;&#xC5D0; &#xB2E8;&#xBC31;&#xC9C8; &#xAE30;&#xB2A5;&#xC744; &#xBE44;&#xD65C;&#xC131;&#xD654; &#xD558;&#xB824;&#xBA74; &#xC0AC;&#xC6A9; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;&#xB294; &#xACF5;&#xAC04;&#xC801; &#xBC0F; fluorophore&#xB97C; &#xC774;&#xC6A9;&#xD55C; &#xAC00;&#xBCBC;&#xC6B4; &#xBE44;&#xD65C;&#xC131;&#xD654; (FALI) &#xB610;&#xB294; &#xBC1C; &#xBCF4;&#xC870; &#xC870;&#xBA85; &#xBE44;&#xD65C;&#xC131;&#xD654; (&#xCE98; &#xB9AC;) &#xC774;&#xB77C;&#xB294; &#xAE30;&#xC220;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC77C;&#xC2DC;&#xC801;&#xC73C;&#xB85C; &#xC815;&#xC758; &#xB41C; &#xBC29;&#xC2DD;&#xC73C;&#xB85C;. &#xC774; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; &alpha;-bungarotoxin &#xBC14;&#xC778;&#xB529; &#xC21C;&#xC11C; (BBS) serotonin&#x2083; A &#xC18C; &#xB2E8;&#xC704; &#xD0DC;&#xADF8; &#xD558; &#xACE0; &#xC774;&#xD6C4;&#xC5D0; &#xB77C;&#xC774;&#xBE0C; &#xC140;&#xC5D0; &#xB193;&#xC5EC;&#xC788;&#xB294;&#xC9C0; &#xD65C;&#xC6A9;&#xB41C; &alpha;-bungarotoxin&#xC640; &#xD568;&#xAED8; 5-HT&#x2083;A/&#xAC8C;&#xC2DC;&#xD310; &#xC218;&#xC6A9; &#xCCB4;&#xB97C; &#xBD84;&#xB958; &#xD569;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xB294; 5-HT&#x2083;A/&#xAC8C;&#xC2DC;&#xD310; &#xC218;&#xC6A9; &#xCCB4; &#xAE38; &#xD56D; &#xC81C;&#xB294; &#xC5C6;&#xB294; &#xACBD;&#xC6B0; &#xB0B4; &#xBA74; constitutively&#xB294; &#xC218;&#xC6A9; &#xCCB4; &#xAE30;&#xB2A5; &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; &#xAD6D;&#xC81C;&#xD654; &#xD615;&#xAD11;&#xC5D0; &#xC758;&#xD574; &#xC800;&#xD574; &#xD45C;&#xC2DC;. &#xC0B0;&#xC18C; &#xAE09;&#xC9C4;&#xC801;&#xC778; &#xCCAD;&#xC18C;&#xBD80; &#xBC18;&#xC751; &#xC0B0;&#xC18C; &#xC885;&#xC758; &#xCC38;&#xC5EC; &#xC81C;&#xC548; FALI &#xD504;&#xB85C;&#xC138;&#xC2A4; &#xC5F0;&#xB8E8; &#xB41C; &#xAE30;&#xB2A5;&#xACFC; &#xAD6D;&#xC81C;&#xD654;&#xC758; &#xD615;&#xAD11; &#xC720;&#xB3C4; &#xC7A5;&#xC560; &#xAC10;&#xC18C; &#xD588;&#xB2E4;. &#xB610;&#xD55C;, &#xC774;&#xB7EC;&#xD55C; &#xB370;&#xC774;&#xD130; &#xB77C;&#xC774;&#xBE0C; &#xC140; &#xD604;&#xBBF8;&#xACBD; &#xC911; &#xAC15;&#xB82C;&#xD55C; &#xC870;&#xBA85; &#xC2E4;&#xC218;&#xB85C; FALI &#xBC0F; &#xB2E8;&#xBC31;&#xC9C8; &#xC778;&#xC2E0; &#xB9E4;&#xB9E4;&#xC758; &#xC800;&#xD574; &#xB420; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

Fluorophore &#xC7AC;&#xC870;&#xD569; 5 HT&#x2083;A &#xC218;&#xC6A9; &#xCCB4; &#xC81C;&#xC815; &#xAD6D;&#xC81C;&#xD654;&#xC640; &#xAE30;&#xB2A5;&#xC758; &#xC870;&#xBA85; &#xBE44;&#xD65C;&#xC131;&#xD654; (FALI)&#xB97C; &#xC9C0;&#xC6D0; &#xD569;&#xB2C8;&#xB2E4;.

Las prote&#xED;nas fluorescentes y las mol&#xE9;culas son ampliamente utilizados para marcar y visualizar las prote&#xED;nas que resultan en una mejor comprensi&#xF3;n de tr&#xE1;fico de prote&#xED;nas, la localizaci&#xF3;n y funci&#xF3;n. Adem&#xE1;s, las etiquetas fluorescentes se han utilizado tambi&#xE9;n para inactivar funci&#xF3;n de las prote&#xED;nas en una forma espacial y temporalmente definido, utilizando una t&#xE9;cnica conocida como fluor&#xF3;foro asistido por inactivaci&#xF3;n de luz (FALI) o crom&#xF3;foro asistida por inactivaci&#xF3;n de luz (CALI). En este estudio hemos marcado la serotonina &#x2083; de la subunidad &#x3B1; con la secuencia-bungarotoxina vinculante (BBS) y marcado posteriormente 5-HT &#x2083; los receptores A / BBS con fluorescencia conjugada &#x3B1;-bungarotoxina en c&#xE9;lulas vivas. Se demuestra que el 5-HT $ &#x2083; $ receptores Un BBS / internalizados son constitutivamente en ausencia de un agonista y la internalizaci&#xF3;n, as&#xED; como la funci&#xF3;n del receptor son inhibidas por fluorescencia. La interrupci&#xF3;n de la fluorescencia inducida por la internalizaci&#xF3;n de la funci&#xF3;n y se redujo con captadores de radicales de ox&#xED;geno que sugiere la participaci&#xF3;n de especies reactivas del ox&#xED;geno, lo que implica el proceso de FALI. Por otra parte, estos datos sugieren que la luz intensa durante la microscopia de c&#xE9;lulas vivas pueden resultar en FALI inadvertida y la inhibici&#xF3;n de tr&#xE1;fico de prote&#xED;nas.

Fluor&#xF3;foro asistido inactivaci&#xF3;n de luz (FALI) recombinante de la 5-HT &#x2083; A la internalizaci&#xF3;n del receptor y la funci&#xF3;n constitutiva

Although public health campaigns advise pregnant women to abstain from ethanol, drinking during pregnancy is pervasive. Here, we highlight recent studies that have clearly demonstrated long-lasting neurobehavioral deficits in the offspring of laboratory animals exposed to moderate levels of ethanol during development. Alterations in learning, memory, motor coordination, social behavior, and stress responses were identified in these animals. Increased vulnerability to substance abuse was also demonstrated. These behavioral alterations have been associated with impairments in neurotransmitter systems, neuromodulators, and/or synaptic plasticity in several brain regions. With this review we hope to contribute to a better appreciation of the potential effects of developmental exposure to moderate ethanol levels, leading to better interventions aimed at relieving fetal alcohol spectrum disorders.

Does moderate drinking harm the fetal brain? Insights from animal models.

The central amygdala (CeA) has a unique role in integrating stress and the rewarding effects of ethanol (EtOH) and plays a major role in the development of EtOH dependence via signaling of corticotropin-releasing factor (CRF). A recent report by Herman and colleagues (2013) entitled "Novel Subunit-Specific Tonic GABA Currents and Differential Effects of Ethanol in the Central Amygdala of CRF Receptor-1 Reporter Mice" is the first study to investigate inhibitory tonic currents in relation to CRF signaling in the CeA. The findings of that study significantly enhance our understanding of inhibitory tonic currents in the CeA and give insight into how EtOH may differentially affect CRF signaling within the CeA, leading to the development of EtOH dependence. This commentary will focus on the recent findings of Herman and colleagues and will discuss the effects of EtOH on the entire anxiety/emotion circuitry.

Ethanol untangles the amygdala-anxiety circuit through tonic GABA inhibition.

Exposure to ethanol (EtOH) during fetal development can lead to long-lasting alterations, including deficits in fine motor skills and motor learning. Studies suggest that these are, in part, a consequence of cerebellar damage. Cerebellar granule neurons (CGNs) are the gateway of information into the cerebellar cortex. Functionally, CGNs are heavily regulated by phasic and tonic GABAergic inhibition from Golgi cell interneurons; however, the effect of EtOH exposure on the development of GABAergic transmission in immature CGNs has not been investigated. To model EtOH exposure during the 3rd trimester-equivalent of human pregnancy, neonatal pups were exposed intermittently to high levels of vaporized EtOH from postnatal day (P) 2 to P12. This exposure gradually increased pup serum EtOH concentrations (SECs) to ∼60 mM (∼0.28 g/dl) during the 4 h of exposure. EtOH levels gradually decreased to baseline 8 h after the end of exposure. Surprisingly, basal tonic and phasic GABAergic currents in CGNs were not significantly affected by postnatal alcohol exposure (PAE). However, PAE increased δ subunit expression at P28 as detected by immunohistochemical and western blot analyses. Also, electrophysiological studies with an agonist that is highly selective for δ-containing GABA(A) receptors, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol (THIP), showed an increase in THIP-induced tonic current. Behavioral studies of PAE rats did not reveal any deficits in motor coordination, except for a delay in the acquisition of the mid-air righting reflex that was apparent at P15 to P18. These findings demonstrate that repeated intermittent exposure to high levels of EtOH during the equivalent of the last trimester of human pregnancy has significant but relatively subtle effects on motor coordination and GABAergic transmission in CGNs in rats.

Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABA(A) receptor δ subunit in cerebellar granule neurons and delays motor development in rats.

Studies indicate that exposure to ethanol (EtOH) during fetal development damages cerebellar Purkinje cells (PCs). PC proximal dendrites receive glutamatergic input from climbing fibers (CFs) originating at the inferior olive. CF input produces a characteristic response in PCs known as the complex spike (CS). During the first 2 weeks of life in rodents (equivalent to the human third trimester of pregnancy), CF-PC synapses undergo profound refinement. Here, we characterized the impact of EtOH exposure during this period on CF-evoked responses in PCs.

Third trimester-equivalent ethanol exposure does not alter complex spikes and climbing fiber long-term depression in cerebellar Purkinje neurons from juvenile rats.

ArticleTotal : 2	Year
Publication title Cited by 48	2014
Publication title	2024

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Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABA(A) receptor δ subunit in cerebellar granule neurons and delays motor development... Neuropharmacology\| PubMed ID: 24316160	2014
Third trimester-equivalent ethanol exposure does not alter complex spikes and climbing fiber long-term depression in cerebellar Purkinje neurons from juvenile rats. Alcoholism, clinical and experimental research\| PubMed ID: 24689500	2014

Russell A. Morton

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