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Titolo:
Ovine caveolin-1: cDNA cloning, E. coli expression, and association with endothelial nitric oxide synthase
Autore:
Chen, DB; Zangl, AL; Zhao, Q; Markley, JL; Zheng, J; Bird, IM; Magness, R;
Indirizzi:
Univ Wisconsin, Meriter Hosp 7E, Dept Obstet & Gynecol, Perinatal Res Labs, Madison, WI 53715 USA Univ Wisconsin Madison WI USA 53715 natal Res Labs, Madison, WI 53715 USA Univ Wisconsin, Dept Biochem, Madison, WI 53715 USA Univ Wisconsin Madison WI USA 53715 , Dept Biochem, Madison, WI 53715 USA Univ Wisconsin, Dept Anim Sci, Madison, WI 53715 USA Univ Wisconsin Madison WI USA 53715 Dept Anim Sci, Madison, WI 53715 USA
Titolo Testata:
MOLECULAR AND CELLULAR ENDOCRINOLOGY
fascicolo: 1-2, volume: 175, anno: 2001,
pagine: 41 - 56
SICI:
0303-7207(20010425)175:1-2<41:OCCCEC>2.0.ZU;2-Y
Fonte:
ISI
Lingua:
ENG
Soggetto:
ESTROGEN-RECEPTOR-ALPHA; PROTEIN-KINASE-C; TYROSINE PHOSPHORYLATION; MOLECULAR-CLONING; PLASMA-MEMBRANE; UTERINE ARTERY; UP-REGULATION; IN-VIVO; CELLS; ACTIVATION;
Keywords:
caveolin-1; endothelial nitric oxide synthase; uterine artery endothelial cells;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
65
Recensione:
Indirizzi per estratti:
Indirizzo: Chen, DB Univ Wisconsin, Meriter Hosp 7E, Dept Obstet & Gynecol, PerinatalRes Labs, 202 S Pk St, Madison, WI 53715 USA Univ Wisconsin 202 S Pk St Madison WI USA 53715 son, WI 53715 USA
Citazione:
D.B. Chen et al., "Ovine caveolin-1: cDNA cloning, E. coli expression, and association with endothelial nitric oxide synthase", MOL C ENDOC, 175(1-2), 2001, pp. 41-56

Abstract

Caveolin-1 (Cav-1), the principal coat protein of caveolae, plays an obligatory role in regulating the activity of, endothelial nitric oxide (NO) synthase (eNOS). We propose that Cav-1 may be critical to eNOS-NO mediated uterine vasodilatation during pregnancy and estrogen replacement therapy. To test this hypothesis in the sheep model, we isolated the full-length cDNA ofovine Cav-1 (oCav-1) from a Lambda ZAP cDNA library of ovine placental artery endothelial cells. Thirty-two positive oCav-1 clones were recognized bya partial oCav-1 cDNA from this library, of which eight were sequenced. Restriction digestion of these clones revealed that the cDNAs of oCav-1 ranged from similar to 2.1 to 2.7 kb. Not-them analysis of Cav-1 mRNAs in ovine uterine artery endothelial cells (UAEC) showed two transcripts of similar to 2.1 and 2.7 kb, respectively. Immunoreactive Cav-1 protein, but not caveolin-2 or caveolin-3, was detected in UAEC. Sequence analysis revealed that in addition to a 537-bp open reading frame encoding a 178 amino acid oCav-1protein, full-length oCav-1 cDNAs apparently possess a similar to 1.6-2.1 kb 3'-untranslated region. Database searches with oCav-1 cDNA revealed thatthe coding region of mammalian Cav-1 genes is highly conserved. We prepared a recombinant full-length oCav-1 protein in which six consecutive histidine residues were tagged at the end of its COOH-terminus and developed a [His](6)-tagged oCav-1 'pull-down assay' for studying the association of eNOS with Cav-1. Incubation of exogenous [His](6)-tagged oCav-1 with resting UAEC extracts led to the formation of a [His](6)-tagged oCav-1-eNOS complex. In the presence of a synthetic caveolin-scaffolding domain (CSD, aa 82-101) peptide, but not a mutated CSD peptide, [His](6)-lagged oCav-1 associated eNOS was dose (0-10 muM)-dependently inhibited, eNOS association with Cav-1 in UAEC was further confirmed by the facts that eNOS co-immunoprecipitated with Cav-1 and vice versa, and that eNOS co-existed with Cav-1 during the isolation of caveolae membranes. Because dissociation of eNOS from Cav-1 is required for the activation of eNOS, eNOS association with Cav-1 in UAEC suggests an important role of Cav-1 in regulating UAEC production of NO and possibly NO-mediated uterine vasodilatation. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

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Documento generato il 23/09/20 alle ore 13:17:04