Prolonged Residence Time of a Noncovalent Molecular Adapter, β-Cyclodextrin, within the Lumen of Mutant α-Hemolysin Pores

Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the α-hemolysin (αHL) pore, thereby offering a basis for the detection of a variety of organic molecules with αHL as a sensor element. β-Cyclodextrin (βCD) resides in the wild-type αHL pore for several hundred microseconds. The residence time can be extended to several milliseconds by the manipulation of pH and transmembrane potential. Here, we describe mutant homoheptameric αHL pores that are capable of accommodating βCD for tens of seconds. The mutants were obtained by site-directed mutagenesis at position 113, which is a residue that lies near a constriction in the lumen of the transmembrane β barrel, and fall into two classes. Members of the tight-binding class, M113D, M113N, M113V, M113H, M113F and M113Y, bind βCD ∼10(4)-fold more avidly than the remaining αHL pores, including WT-αHL. The lower K (d) values of these mutants are dominated by reduced values of k(off). The major effect of the mutations is most likely a remodeling of the binding site for βCD in the vicinity of position 113. In addition, there is a smaller voltage-sensitive component of the binding, which is also affected by the residue at 113 and may result from transport of the neutral βCD molecule by electroosmotic flow. The mutant pores for which the dwell time of βCD is prolonged can serve as improved components for stochastic sensors

Dominant Cone-Rod Dystrophy: A Mouse Model Generated by Gene Targeting of the GCAP1/Guca1a Gene

Cone dystrophy 3 (COD3) is a severe dominantly inherited retinal degeneration caused by missense mutations in GUCA1A, the gene encoding Guanylate Cyclase Activating Protein 1 (GCAP1). The role of GCAP1 in controlling cyclic nucleotide levels in photoreceptors has largely been elucidated using knock-out mice, but the disease pathology in these mice cannot be extrapolated directly to COD3 as this involves altered, rather than loss of, GCAP1 function. Therefore, in order to evaluate the pathology of this dominant disorder, we have introduced a point mutation into the murine Guca1a gene that causes an E155G amino acid substitution; this is one of the disease-causing mutations found in COD3 patients. Disease progression in this novel mouse model of cone dystrophy was determined by a variety of techniques including electroretinography (ERG), retinal histology, immunohistochemistry and measurement of cGMP levels. It was established that although retinal development was normal up to 3 months of age, there was a subsequent progressive decline in retinal function, with a far greater alteration in cone than rod responses, associated with a corresponding loss of photoreceptors. In addition, we have demonstrated that accumulation of cyclic GMP precedes the observed retinal degeneration and is likely to contribute to the disease mechanism. Importantly, this knock-in mutant mouse has many features in common with the human disease, thereby making it an excellent model to further probe disease pathogenesis and investigate therapeutic interventions

Facteurs de non relever du sol en population gériatrique (Etude de soixante admissions en court et moyen séjour gériatrique)

STRASBOURG-Medecine (674822101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

L'hopital de semaine, nouvelle structure en gériatrie (ses intérêts et ses limites)

STRASBOURG-Medecine (674822101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Végétation des étangs de la Brenne (Indre) : influence des pratiques piscicoles à l'échelle des communautés végétales et sur une espèce d'intérêt européen : Caldesia parnassifolia (L.) Parl.

PARIS-Museum Hist.Naturelle (751052304) / SudocSudocFranceF

Variations temporelles des populations de Caldesia parnassifloia (L.) Parlatore dans les étangs de la Brenne area (Indre, France)

National audienc