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1.
Am J Physiol Renal Physiol ; 311(2): F411-23, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27306979

RESUMO

The antidiuretic hormone vasopressin (AVP) regulates renal salt and water reabsorption along the distal nephron and collecting duct system. These effects are mediated by vasopressin 2 receptors (V2R) and release of intracellular Gs-mediated cAMP to activate epithelial transport proteins. Inactivating mutations in the V2R gene lead to the X-linked form of nephrogenic diabetes insipidus (NDI), which has chiefly been related with impaired aquaporin 2-mediated water reabsorption in the collecting ducts. Previous work also suggested the AVP-V2R-mediated activation of Na(+)-K(+)-2Cl(-)-cotransporters (NKCC2) along the thick ascending limb (TAL) in the context of urine concentration, but its individual contribution to NDI or, more generally, to overall renal function was unclear. We hypothesized that V2R-mediated effects in TAL essentially determine its reabsorptive function. To test this, we reevaluated V2R expression. Basolateral membranes of medullary and cortical TAL were clearly stained, whereas cells of the macula densa were unreactive. A dominant-negative, NDI-causing truncated V2R mutant (Ni3-Glu242stop) was then introduced into the rat genome under control of the Tamm-Horsfall protein promoter to cause a tissue-specific AVP-signaling defect exclusively in TAL. Resulting Ni3-V2R transgenic rats revealed decreased basolateral but increased intracellular V2R signal in TAL epithelia, suggesting impaired trafficking of the receptor. Rats displayed significant baseline polyuria, failure to concentrate the urine in response to water deprivation, and hypercalciuria. NKCC2 abundance, phosphorylation, and surface expression were markedly decreased. In summary, these data indicate that suppression of AVP-V2R signaling in TAL causes major impairment in renal fluid and electrolyte handling. Our results may have clinical implications.


Assuntos
Rim/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Vasopressinas/genética , Vasopressinas/fisiologia , Animais , Proteínas de Transporte/metabolismo , AMP Cíclico/metabolismo , Ciclo-Oxigenase 2/metabolismo , Diabetes Insípido Nefrogênico/genética , Epitélio/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/genética , Sistema Justaglomerular/metabolismo , Rim/ultraestrutura , Córtex Renal/metabolismo , Medula Renal/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Receptores de Vasopressinas/genética , Membro 1 da Família 12 de Carreador de Soluto/genética , Membro 1 da Família 12 de Carreador de Soluto/metabolismo
3.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 63(Pt 10): 839-43, 2007 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-17909284

RESUMO

Chemically synthesized RNAs with the unnatural L-configuration possess enhanced in vivo stability and nuclease resistance, which is a highly desirable property for pharmacological applications. For a structural comparison, both L- and D-RNA oligonucleotides of a shortened Thermus flavus 5S rRNA A-helix were chemically synthesized. The enantiomeric RNA duplexes were stochiometrically cocrystallized as a racemate, which enabled analysis of the D- and L-RNA enantiomers in the same crystals. In addition to a biochemical investigation, diffraction data were collected to 3.0 A resolution using synchrotron radiation. The crystals belonged to space group P3(1)21, with unit-cell parameters a = b = 35.59, c = 135.30 A, gamma = 120 degrees and two molecules per asymmetric unit.


Assuntos
RNA Ribossômico 5S/análise , RNA Ribossômico 5S/química , Difração de Raios X/métodos , Cristalização , Estrutura Secundária de Proteína/fisiologia , Estereoisomerismo
4.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 63(Pt 10): 858-61, 2007 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-17909289

RESUMO

The major dissimilarities between the eukaryotic/archaebacterial-type and eubacterial-type glycyl-tRNA synthetase systems (GlyRS; class II aminoacyl-tRNA synthetases) represent an intriguing example of evolutionarily divergent solutions to similar biological functions. The differences in the identity elements of the respective tRNA(Gly) systems are located within the acceptor stem and include the discriminator base U73. In the present work, the human tRNA(Gly) acceptor-stem microhelix was crystallized in an attempt to analyze the structural features that govern the correct recognition of tRNA(Gly) by the eukaryotic/archaebacterial-type glycyl-tRNA synthetase. The crystals of the human tRNA(Gly) acceptor-stem helix belong to the monoclinic space group C2, with unit-cell parameters a = 37.12, b = 37.49, c = 30.38 A, alpha = gamma = 90, beta = 113.02 degrees, and contain one molecule per asymmetric unit. A high-resolution data set was acquired using synchrotron radiation and the data were processed to 1.2 A resolution.


Assuntos
RNA de Transferência de Glicina/química , RNA de Transferência de Glicina/isolamento & purificação , Cristalização , Cristalografia por Raios X , Humanos , Conformação de Ácido Nucleico , RNA de Transferência de Glicina/genética
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