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81.
María Isabel Niemeyer L. Pablo Cid Gaspar Pe?a-Münzenmayer Francisco V. Sepúlveda 《The Journal of biological chemistry》2010,285(22):16467-16475
TASK-2 (KCNK5 or K2P5.1) is a background K+ channel that is opened by extracellular alkalinization and plays a role in renal bicarbonate reabsorption and central chemoreception. Here, we demonstrate that in addition to its regulation by extracellular protons (pHo) TASK-2 is gated open by intracellular alkalinization. The following pieces of evidence suggest that the gating process controlled by intracellular pH (pHi) is independent from that under the command of pHo. It was not possible to overcome closure by extracellular acidification by means of intracellular alkalinization. The mutant TASK-2-R224A that lacks sensitivity to pHo had normal pHi-dependent gating. Increasing extracellular K+ concentration acid shifts pHo activity curve of TASK-2 yet did not affect pHi gating of TASK-2. pHo modulation of TASK-2 is voltage-dependent, whereas pHi gating was not altered by membrane potential. These results suggest that pHo, which controls a selectivity filter external gate, and pHi act at different gating processes to open and close TASK-2 channels. We speculate that pHi regulates an inner gate. We demonstrate that neutralization of a lysine residue (Lys245) located at the C-terminal end of transmembrane domain 4 by mutation to alanine abolishes gating by pHi. We postulate that this lysine acts as an intracellular pH sensor as its mutation to histidine acid-shifts the pHi-dependence curve of TASK-2 as expected from its lower pKa. We conclude that intracellular pH, together with pHo, is a critical determinant of TASK-2 activity and therefore of its physiological function. 相似文献
82.
Maria Rosário Sambo Maria Jesus Trovoada Carla Benchimol Vatúsia Quinhentos Lígia Gon?alves Rute Velosa Maria Isabel Marques Nuno Sepúlveda Taane G. Clark Stefan Mustafa Oswald Wagner António Coutinho Carlos Penha-Gon?alves 《PloS one》2010,5(6)
Background
Cerebral malaria (CM) represents a severe outcome of the Plasmodium falciparum infection. Recent genetic studies have correlated human genes with severe malaria susceptibility, but there is little data on genetic variants that increase the risk of developing specific malaria clinical complications. Nevertheless, susceptibility to experimental CM in the mouse has been linked to host genes including Transforming Growth Factor Beta 2 (TGFB2) and Heme oxygenase-1 (HMOX1). Here, we tested whether those genes were governing the risk of progressing to CM in patients with severe malaria syndromes.Methodology/Principal Findings
We report that the clinical outcome of P. falciparum infection in a cohort of Angolan children (n = 430) correlated with nine TGFB2 SNPs that modify the risk of progression to CM as compared to other severe forms of malaria. This genetic effect was explained by two haplotypes harboring the CM-associated SNPs (Pcorrec. = 0.035 and 0.036). In addition, one HMOX1 haplotype composed of five CM-associated SNPs increased the risk of developing the CM syndrome (Pcorrec. = 0.002) and was under-transmitted to children with uncomplicated malaria (P = 0.036). Notably, the HMOX1-associated haplotype conferred increased HMOX1 mRNA expression in peripheral blood cells of CM patients (P = 0.012).Conclusions/Significance
These results represent the first report on CM genetic risk factors in Angolan children and suggest the novel hypothesis that genetic variants of the TGFB2 and HMOX1 genes may contribute to confer a specific risk of developing the CM syndrome in patients with severe P. falciparum malaria. This work may provide motivation for future studies aiming to replicate our findings in larger populations and to confirm a role for these genes in determining the clinical course of malaria. 相似文献83.
Debra McLaggan Noppadon Adjimatera Kristina Sepčić Marcel Jaspars David J MacEwan Ian S Blagbrough Roderick H Scott 《BMC biotechnology》2006,6(1):6-12
Background
Haplosclerid marine sponges produce pore forming polyalkylpyridinium salts (poly-APS), which can be used to deliver macromolecules into cells. The aim of this study was to investigate the delivery of DNA, siRNA and lucifer yellow into cells mediated by poly-APS and its potential mechanisms as compared with other lipofection systems (lipofectamine and N 4,N 9-dioleoylspermine (LipoGen)). DNA condensation was evaluated and HEK 293 and HtTA HeLa cells were used to investigate pore formation and intracellular delivery of cDNA, siRNA and lucifer yellow. 相似文献84.
Valbuena JJ Bravo RV Ocampo M Lopez R Rodriguez LE Curtidor H Puentes A Garcia JE Tovar D Gomez J Leiton J Patarroyo ME 《Biochemical and biophysical research communications》2004,321(4):835-844
Erythrocyte binding antigen-160 (EBA-160) protein is a Plasmodium falciparum antigen homologue from the erythrocyte binding protein family (EBP). It has been shown that the EBP family plays a role in parasite binding to the erythrocyte surface. The EBA-160 sequence has been chemically synthesised in seventy 20-mer sequential peptides covering the entire 3D7 protein strain, each of which was tested in erythrocyte binding assays to identify possible EBA-160 functional regions. Five EBA-160 high activity binding peptides (HABPs) specifically binding to erythrocytes with high affinity were identified. Dissociation constants lay between 200 and 460 nM and Hill coefficients between 1.5 and 2.3. Erythrocyte membrane protein binding peptide cross-linking assays using SDS-PAGE showed that these peptides bound specifically to 12, 28, and 44 kDa erythrocyte membrane proteins. The nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. HABPs were able to block merozoite in vitro invasion of erythrocytes. HABPs’ potential as anti-malarial vaccine candidates is also discussed. 相似文献
85.
Thioridazine inhibits the activity of the synaptic plasma membrane Ca(2+)-ATPase from pig brain and slightly decreases the rate of Ca(2+) accumulation by synaptic plasma membrane vesicles in the absence of phosphate. However, in the presence of phosphate, thioridazine increases the rate of Ca(2+) accumulation into synaptic plasma membrane vesicles. Phosphate anions diffuse through the membrane and form calcium phosphate crystals, reducing the free Ca(2+) concentration inside the vesicles and the rate of Ca(2+) leak. The higher levels of Ca(2+) accumulation obtained in the presence of thioridazine could be explained by a reduction of the rate of slippage on the plasma membrane ATPase. 相似文献
86.
87.
88.
Acosta-Rivero N Rodriguez A Mussachio A Poutou J Falcon V Torres D Aguilar JC Linares M Alonso M Perez A Menendez I Morales-Grillo J Marquez G Dueñas-Carrera S 《Biochemical and biophysical research communications》2005,334(3):901-906
Little is known about the assembly pathway or structure of the hepatitis C virus (HCV). In this work a truncated HCcAg variant covering the first 120 aa (HCcAg.120) with a 32 aa N-terminal fusion peptide (6x Histag-Xpress epitope) was purified as a monomer under strong denaturing conditions. In addition, minor HCcAg.120 peaks exhibiting little different molecular mass by SDS-PAGE which possibly represents alternative forms harboring the N-termini of HCcAg.120 were detected. Analysis using gel filtration chromatography showed that HCcAg.120 assembled into high molecular weight structures in vitro in the absence of structured nucleic acids. The negative-stain electron microscopy analysis revealed that these structures correspond with spherical VLPs of uniform morphology and size distribution. The diameters of these particles ranged from 20 to 43nm with an average diameter of approximately 30 nm and were specifically immunolabelled with a mouse monoclonal antibody against the residues 5-35 of HCcAg. Results presented in this work showed that HCcAg.120 assembled in vitro into VLPs in the absence of structured nucleic acids with similar morphology and size distribution to those found in sera and hepatocytes from HCV-infected patients. Therefore, these VLPs would be important to elucidate the mechanisms behind the ability of HCcAg to assemble into a nucleocapsid structure. 相似文献
89.
Splice variants of a ClC-2 chloride channel with differing functional characteristics 总被引:8,自引:0,他引:8
Cid LP Niemeyer MI Ramírez A Sepúlveda FV 《American journal of physiology. Cell physiology》2000,279(4):C1198-C1210
We identified two ClC-2 clones in a guinea pigintestinal epithelial cDNA library, one of which carries a 30-bpdeletion in the NH2 terminus. PCR using primersencompassing the deletion gave two products that furthermore wereamplified with specific primers confirming their authenticity. Thecorresponding genomic DNA sequence gave a structure of three exons andtwo introns. An internal donor site occurring within one of the exonsaccounts for the deletion, consistent with alternative splicing.Expression of the variants gpClC-2 and gpClC-277-86 in HEK-293cells generated inwardly rectifying chloride currents with similaractivation characteristics. Deactivation, however, occurred with fasterkinetics in gpClC-277-86. Site-directed mutagenesis suggeststhat a protein kinase C-mediated phosphorylation consensus site lost ingpClC-277-86 is not responsible for the observed change. Thedeletion-carrying variant is found in most tissues examined, and itappears more abundant in proximal colon, kidney, and testis. Thepresence of a splice variant of ClC-2 modified in itsNH2-terminal domain could have functional consequences intissues where their relative expression levels are different. 相似文献
90.