Cysteine-terminated B-domain of Staphylococcus aureus protein A as a scaffold for targeting GABA(A) receptors.

This study reports the preparation and characterization of cysteine-terminated B-domain (Bd-cys) of Staphylococcus aureus protein A, in combination with immunoglobulin G (IgG) antibodies directed against the ρ1 and α1 subunits of GABA(A) receptors, for localizing reagents of interest to the target receptor. A cysteine residue was inserted at the C terminus of the cysteine-lacking B-domain (Bd) and used for conjugating maleimide-containing compounds. As determined by enzyme-linked immunosorbent assay (ELISA), binding of a Bd-cys-S-fluorescein conjugate to polyclonal guinea pig anti-GABA(A)-ρ1 and rabbit anti-GABA(A)-α1 IgG was similar to that exhibited by full-length protein A.

Recovery of rod photoresponses in ABCR-deficient mice.

Purpose: The ABCR protein of the rod outer segment is thought to facilitate movement of the all-trans retinal photoproduct of rhodopsin bleaching out of the disc lumen. This study was undertaken to investigate the extent to which ABCR deficiency affects the post-bleach recovery of the rod photoresponse in ABCR-deficient (abcr-/-) mice.

Methods: Electroretinographic (ERG) a-wave responses were recorded from abcr-/- mice and two control strains.

Preservation of retinoid influx into eye tissues of ABCR-deficient mice.

Using an in vivo radiolabeling technique, we investigated the movement of retinoid into the retinal pigment epithelium (RPE) of the abcr-/- mouse, which lacks the photoreceptor ABCR protein and is a model for Stargardt disease. Eye tissues and serum obtained from dark-adapted, 5- to 8-month-old abcr-/- and control mice following the intraperitoneal injection of all-trans ((3)H)retinol were analyzed to determine the inferred influx of retinoid from the serum into the RPE. At 4.

Activation of membrane receptors by neurotransmitter released from temperature-sensitive hydrogels.

The present paper describes the design, construction and testing of a temperature-sensitive N-isopropylacrylamide hydrogel device for studying the controlled presentation of gamma-aminobutyric acid (GABA) to GABA(C) membrane receptors expressed in Xenopus laevis oocytes. Upon temperature lowering, the GABA-loaded hydrogel positioned near the surface of the GABA(C)-expressing oocyte elicits a membrane current response resembling that induced by superfusion of the oocyte with free GABA. The response to cooling is not observed when GABA is omitted from the hydrogel loading solution.

Interphotoreceptor retinoid-binding protein (IRBP) promotes the release of all-trans retinol from the isolated retina following rhodopsin bleaching illumination.

All-trans retinol generated in rod photoreceptors upon the bleaching of rhodopsin is known to move from the rods to the retinal pigment epithelium (RPE), where it is enzymatically converted to 11-cis retinal in the retinoid visual cycle. Interphotoreceptor retinoid-binding protein (IRBP) contained in the extracellular compartment (interphotoreceptor matrix) that separates the retina and RPE has been hypothesized to facilitate this movement of all-trans retinol, but the precise role of IRBP in this process remains unclear. To examine the activity of IRBP in the release of all-trans retinol from the rods, initially dark-adapted isolated retinas obtained from toad (Bufo marinus) eyes were bleached and then incubated in darkness for defined periods (5-180 min) in physiological saline (Ringer solution) supplemented with IRBP (here termed 'IRBP I') at defined concentrations (2-90 microm).

Features of visual function in the naked mole-rat Heterocephalus glaber.

The eyes and visual capacity of the naked mole-rat, Heterocephalus glaber, a subterranean rodent, were evaluated using anatomical, biochemical, and functional assays, and compared to other rodents of similar body size (mouse and gerbil). The eye is small compared to mouse, yet possesses cornea, lens, and retina with typical mammalian organization. The optic nerve cross-sectional area and fiber density are approximately 10% and approximately 50% that of gerbil, respectively.

Acute radiolabeling of retinoids in eye tissues of normal and rpe65-deficient mice.

Purpose: Mice with a targeted disruption of the gene encoding RPE65, a protein ordinarily highly expressed in the retinal pigment epithelium (RPE), accumulate abnormally high levels of all-trans retinyl ester in the RPE and exhibit very little 11-cis retinal in the retina. The present study was undertaken to determine whether the Rpe65-deficient mouse exhibits an abnormal flux of retinoid between the systemic circulation and the eye tissues.

Methods: Dark-adapted Rpe65-deficient mice (Rpe65(-/-)) and wild-type control mice (Rpe65(+/+)) of approximate ages 1 and 3 months received an intraperitoneal injection of all-trans ((3)H)retinol.

Excitation and desensitization of mouse rod photoreceptors in vivo following bright adapting light.

Electroretinographic (ERG) methods were used to determine response properties of mouse rod photoreceptors in vivo following adapting illumination that produced a significant extent of rhodopsin bleaching. Bleaching levels prevailing at approximately 10 min and approximately 20 min after the adapting exposure were on average 14 % and 9 %, respectively, based on the analysis of visual cycle retinoids in the eye tissues. Recovery of the rod response to the adapting light was monitored by analysing the ERG a-wave response to a bright probe flash presented at varying times during dark adaptation.