PubMed Health⌕ Search

Biomedical subjects

N V Krasnoperova

Publications and source records attributed to N V Krasnoperova.

6 recordsLinked to original sources
PubMed2002

Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice.

A 10 microm spot of argon laser light was focused onto the outer segments of intact mouse rods loaded with fluo-3, fluo-4 or fluo-5F, to estimate dark, resting free Ca(2+) concentration ([Ca(2+)](i)) and changes in [Ca(2+)](i) upon illumination. Dye concentration was adjusted to preserve the normal physiology of the rod, and the laser intensity was selected to minimise bleaching of the fluorescent dye. Wild-type mouse rods illuminated continuously with laser light showed a progressive decrease in fluorescence well fitted by two exponentials with mean time constants of 154 and 540 ms. Rods from transducin alpha-subunit knock-out (Tralpha-/-) animals showed no light-dependent decline in fluorescence but exhibited an initial rapid component of fluorescence increase which could be fitted with a single exponential (tau~1-4 ms). This fluorescence increase was triggered by rhodopsin bleaching, since its amplitude was reduced by pre-exposure to bright bleaching light and its time constant decreased with increasing laser intensity. The rapid component was however unaffected by incorporation of the calcium chelator BAPTA and seemed therefore not to reflect an actual increase in [Ca(2+)](i). A similar rapid increase in fluorescence was also seen in the rods of wild-type mice just preceding the fall in fluorescence produced by the light-dependent decrease in [Ca(2+)](i). Dissociation constants were measured in vitro for fluo-3, fluo-4 and fluo-5F with and without 1 mM Mg(2+) from 20 to 37 degrees C. All three dyes showed a strong temperature dependence, with the dissociation constant changing by a factor of 3-4 over this range. Values at 37 degrees C were used to estimate absolute levels of rod [Ca(2+)](i). All three dyes gave similar values for [Ca(2+)](i) in wild-type rods of 250 +/- 20 nM in darkness and 23 +/- 2 nM after exposure to saturating light. There was no significant difference in dark [Ca(2+)](i) between wild-type and Tralpha-/- animals.

Animals↗
PubMed2001

AAV-mediated delivery of ciliary neurotrophic factor prolongs photoreceptor survival in the rhodopsin knockout mouse.

Retinitis pigmentosa (RP), an inherited retinal degenerative disease causing blindness, is characterized by progressive apoptotic death of photoreceptors. Therapeutic modification of photoreceptor apoptosis may provide an effective therapy for this disorder. Ciliary neurotrophic factor (CNTF) has been shown to promote survival of a number of different neuronal cell types, including photoreceptors. The present study aimed to test whether adeno-associated virus (AAV)-mediated delivery of the gene encoding CNTF delays photoreceptor death in the rhodopsin knockout (opsin(-/-)) mouse, an animal model of RP. The vector was made to express a secretable form of CNTF in tandem with a marker GFP. Cultured 293 cells transduced with this virus expressed both CNTF and GFP. The conditioned media from such cells supported the survival of chick dorsal root ganglion neurons in the same manner as recombinant CNTF. Subretinal administration of this virus led to efficient transduction of photoreceptors as indicated by GFP fluorescence and CNTF immunostaining. Histologic examination showed significant photoreceptor preservation in the injected quadrant of the retina. This protection lasted through termination of the experiment (3 months). AAV-mediated delivery of CNTF may have implications for the treatment of human retinal degeneration.

Animals↗
PubMed2001

Mutant rhodopsin transgene expression on a null background.

PURPOSE: To study mechanisms leading to photoreceptor degeneration in mouse models for autosomal dominant retinitis pigmentosa (adRP) based on the rhodopsin P23H mutation. METHODS: Mice of a transgenic line expressing a rhodopsin triple mutant, V20G, P23H, and P27L (GHL), were mated with rhodopsin (rho) knockout mice. Littermates of various ages and genotypes (GHL+rho+/+, GHL+rho+/-, and GHL+rho-/-) were examined for outer nuclear layer thickness and outer segment formation (histology), fate of mutant rhodopsin (immunocytochemistry), and photoreceptor function (electroretinogram; ERG). RESULTS: Mice expressing GHL-rhodopsin in the absence of wild-type rhodopsin had severe retinopathy, which was nearly complete by postnatal day (P)30. GHL-rhodopsin formed homodimers nearly exclusively on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, whereas wild-type rhodopsin predominantly formed monomers. Expression level of mutant rhodopsin in predegenerate (P10) GHL+rho-/- retinas was low, approximately 10% to 25% of normal levels. No elaboration of disc membrane or outer segment formation was observed at any time point examined. The mutant rhodopsin was found mostly in perinuclear locales (endoplasmic reticulum; ER) as evidenced by colocalization using the antibodies Rho1D4 and calnexin-NT. CONCLUSIONS: GHL-rhodopsin dimerizes, localizes to the ER, and fails to transport and support outer segment formation. Additionally, the mutant protein does not support a scotopic ERG a-wave and accelerates photoreceptor degeneration over that occurring with the rhodopsin knockout alone. These findings indicate a cytotoxic effect of the mutant protein, probably elicited by an unfolded protein response.

Animals↗
PubMed2000

Phototransduction in transgenic mice after targeted deletion of the rod transducin alpha -subunit.

Retinal photoreceptors use the heterotrimeric G protein transducin to couple rhodopsin to a biochemical cascade that underlies the electrical photoresponse. Several isoforms of each transducin subunit are present in the retina. Although rods and cones seem to contain distinct transducin subunits, it is not known whether phototransduction in a given cell type depends strictly on a single form of each subunit. To approach this question, we have deleted the gene for the rod transducin alpha-subunit in mice. In hemizygous knockout mice, there was a small reduction in retinal transducin alpha-subunit content but retinal morphology and the physiology of single rods were largely normal. In homozygous knockout mice, a mild retinal degeneration occurred with age. Rod-driven components were absent from the electroretinogram, whereas cone-driven components were retained. Every photoreceptor examined by single-cell recording failed to respond to flashes, with one exception. The solitary responsive cell was insensitive, as expected for a cone, but had a rod-like spectral sensitivity and flash response kinetics that were slow, even for rods. These results indicate that most if not all rods use a single transducin type in phototransduction.

Animals↗
PubMed1999

Morphological, physiological, and biochemical changes in rhodopsin knockout mice.

Mutations in rod opsin, the visual pigment protein of rod photoreceptors, account for approximately 15% of all inherited human retinal degenerations. However, the physiological and molecular events underlying the disease process are not well understood. One approach to this question has been to study transgenic mice expressing opsin genes containing defined mutations. A caveat of this approach is that even the overexpression of normal opsin leads to photoreceptor cell degeneration. To overcome the problem, we have reduced or eliminated endogenous rod opsin content by targeted gene disruption. Retinas in mice lacking both opsin alleles initially developed normally, except that rod outer segments failed to form. Within months of birth, photoreceptor cells degenerated completely. Retinas from mice with a single copy of the opsin gene developed normally, and rods elaborated outer segments of normal size but with half the normal complement of rhodopsin. Photoreceptor cells in these retinas also degenerated but did so over a much slower time course. Physiological and biochemical experiments showed that rods from mice with a single opsin gene were approximately 50% less sensitive to light, had accelerated flash-response kinetics, and contained approximately 50% more phosducin than wild-type controls.

Animals↗