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3.
Plant J ; 52(3): 449-59, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17764516

RESUMO

The vacuole represents a pivotal plant organelle for management of ion homeostasis, storage of proteins and solutes, as well as deposition of cytotoxic compounds. Ion channels, pumps and carriers in the vacuolar membrane under control of cytosolic factors provide for ionic and metabolic homeostasis between this storage organelle and the cytoplasm. Here we show that AtTPK1 (KCO1), a vacuolar membrane localized K(+) channel of the TPK family, interacts with 14-3-3 proteins (general regulating factors, GRFs). Following in planta expression TPK1 and GRF6 co-localize at the vacuolar membrane. Co-localization of wild-type TPK1, but not the TPK1-S42A mutant, indicates that phosphorylation of the 14-3-3 binding motif of TPK1 represents a prerequisite for interaction. Pull-down assays and surface plasmon resonance measurements revealed GRF6 high-affinity interaction with TPK1. Following expression of TPK1 in yeast and isolation of vacuoles, patch-clamp studies identified TPK1 as a voltage-independent and Ca(2+)-activated K(+) channel. Addition of 14-3-3 proteins strongly increased the TPK1 activity in a dose-dependent manner. However, an inverse effect of GRF6 on the activity of the slow-activating vacuolar (SV) channel was observed in mesophyll vacuoles from Arabidopsis thaliana. Thus, TPK1 seems to provide for a Ca(2+)- and 14-3-3-sensitive mechanism capable of controlling cytoplasmic potassium homeostasis in plants.


Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Vacúolos/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/isolamento & purificação , Sítios de Ligação , Cálcio/metabolismo , Regulação para Baixo , Membranas Intracelulares/metabolismo , Dados de Sequência Molecular , Fosforilação , Canais de Potássio de Domínios Poros em Tandem/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
4.
Proc Natl Acad Sci U S A ; 101(44): 15621-6, 2004 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-15505206

RESUMO

The Arabidopsis tandem-pore K(+) (TPK) channels displaying four transmembrane domains and two pore regions share structural homologies with their animal counterparts of the KCNK family. In contrast to the Shaker-like Arabidopsis channels (six transmembrane domains/one pore region), the functional properties and the biological role of plant TPK channels have not been elucidated yet. Here, we show that AtTPK4 (KCO4) localizes to the plasma membrane and is predominantly expressed in pollen. AtTPK4 (KCO4) resembles the electrical properties of a voltage-independent K(+) channel after expression in Xenopus oocytes and yeast. Hyperpolarizing as well as depolarizing membrane voltages elicited instantaneous K(+) currents, which were blocked by extracellular calcium and cytoplasmic protons. Functional complementation assays using a K(+) transport-deficient yeast confirmed the biophysical and pharmacological properties of the AtTPK4 channel. The features of AtTPK4 point toward a role in potassium homeostasis and membrane voltage control of the growing pollen tube. Thus, AtTPK4 represents a member of plant tandem-pore-K(+) channels, resembling the characteristics of its animal counterparts as well as plant-specific features with respect to modulation of channel activity by acidosis and calcium.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cálcio/metabolismo , Membrana Celular/metabolismo , Feminino , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Potenciais da Membrana , Dados de Sequência Molecular , Mutação , Oócitos/metabolismo , Pólen/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Xenopus
5.
Eur J Anaesthesiol ; 17(10): 634-41, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11050522

RESUMO

In a double-blind randomized study, the incidence and severity of postoperative nausea and vomiting was investigated with a new formulation of etomidate (Etomidate-(R)Lipuro, B. Braun Melsungen AG, Germany) compared with propofol for induction of a balanced anaesthesia with isoflurane/fentanyl in air. The incidence and intensity of nausea was examined by use of a visual analogue scale (VAS; 0-100 mm) at 1, 2, between 6 and 8, and 24 h postoperatively. One-hundred-and-sixty-four patients undergoing orthopedic procedures were studied. For etomidate vs. propofol, 14.6% vs. 14.2% male and 26.8% vs. 27.5% female patients were nauseated during the first two postoperative hours. The median rating for nausea remained below 5 mm at any time in both groups, i.e. the intensity of nausea was very low. The incidence of vomiting was higher in women receiving etomidate (26.8% vs. 10%). We conclude that etomidate does not increase nausea during the early postoperative period.


Assuntos
Anestésicos Combinados , Anestésicos Intravenosos/efeitos adversos , Etomidato/efeitos adversos , Náusea e Vômito Pós-Operatórios/induzido quimicamente , Propofol/administração & dosagem , Adulto , Anestésicos Intravenosos/administração & dosagem , Método Duplo-Cego , Etomidato/administração & dosagem , Emulsões Gordurosas Intravenosas , Feminino , Humanos , Masculino , Estudos Prospectivos
6.
Nucleosides Nucleotides Nucleic Acids ; 19(4): 749-56, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10960033

RESUMO

The phosphodiester bond between U4 and G5 in the U-turn of a chemically modified hammerhead ribozyme was substituted by an amide backbone without compromising the ribozyme's cleavage activity. Furthermore, the modified ribozyme proved to be completely stable against endonucleolytic digestion at this position.


Assuntos
Amidas/química , Guanidina/análogos & derivados , RNA Catalítico/química , RNA Catalítico/metabolismo , Uridina/análogos & derivados , Sítios de Ligação , Catálise , Endonucleases/metabolismo , Guanidina/síntese química , Modelos Moleculares , Conformação de Ácido Nucleico , Relação Estrutura-Atividade , Uridina/síntese química
7.
Bioorg Med Chem Lett ; 9(5): 787-92, 1999 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-10201848

RESUMO

The 2'-C-difluoromethylated nucleoside 4 was synthesized starting from uridine. 4 was then converted to the 3'-O-phosphoramidite derivative 5 and was incorporated into a hammerhead ribozyme (7). The cleavage characteristics of the modified oligonucleotide have been analysed.


Assuntos
RNA Catalítico/efeitos dos fármacos , Uridina/análogos & derivados , Animais , Sequência de Bases , Catálise , Estabilidade Enzimática/efeitos dos fármacos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Catalítico/metabolismo , Tubarões , Uridina/síntese química , Uridina/farmacologia
8.
J Pharmacol Exp Ther ; 278(3): 1419-27, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8819529

RESUMO

Modulation of gene expression via nucleic acid sequence-specific intervention represents a new paradigm for drug discovery and development. Ribozymes are small RNA structures capable of cleaving RNA target molecules in a catalytic fashion. A 2'-O-allyl-modified hammerhead ribozyme designed to cleave the messenger RNA of cytochrome P-450 3A2 was administered to rats via 0.25 mg intravenous injections to investigate the disposition of this compound. The chemically modified ribozyme binds to serum albumin and can be displaced by phosphorothioate oligonucleotides. A biphasic plasma clearance with a distribution half-life of 12 min and an elimination half-life of 6.5 h was observed. A volume of distribution of 2.1 l/kg indicates perfusion into tissues well beyond the vascular system. The chemically modified ribozyme can be detected intact in the plasma up to 48 h after injection. Metabolic degradation of the chemically modified ribozyme occurs at unmodified ribonucleotides, leaving the 2'-O-allyl-modified sites intact. Recovery of intact chemically modified ribozyme was 1.9% of the administered dose at 12 h along with significant metabolites. The renal clearance of the intact ribozyme is an average 34.3 ml/h. The tissue distribution of the chemically modified ribozyme at 48 h is primarily to kidney and liver but the only detected material is a single 27-mer metabolite that has been cut in the unmodified GAAA region. The brain concentration of the prominent 27-mer metabolite is greater than that observed in the lung or spleen. Examination of tissues reveals no morphological evidence of toxicity. These data strongly support the potential utility of synthetic, 2'-O-allyl-modified hammerhead ribozymes as therapeutic agents in vivo.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , RNA Catalítico/farmacocinética , Esteroide Hidroxilases/genética , Animais , Injeções Intravenosas , Fígado/metabolismo , Masculino , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Albumina Sérica/metabolismo , Relação Estrutura-Atividade , Distribuição Tecidual
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