[Show abstract][Hide abstract] ABSTRACT: To generate and characterize a constitutively active, RPE-specific, cre-expressing transgenic mouse line. This line can be used to create RPE-specific knockouts by crossing with mice harboring loxP-flanked (floxed) genes.
A transgene construct was assembled with the BEST1 promoter driving cre expression. Transgenic mice were generated on a C57BL/6 background. Cre expression was assessed by immunofluorescence and Western blot analysis. Cre enzymatic activity was tested by crossing to three lines with floxed DNA regions and detecting deletion of the intervening sequences or through histochemical detection of lacZ activity. Potential cre-mediated toxicity was assessed by retinal histology up to 24 months of age and by electroretinography.
The BEST1-cre line with expression in the highest percentage of RPE cells displayed a patchy mosaic expression pattern, with 50% to 90% of RPE cells expressing cre. In mice outcrossed to a mixed B6/129 background, expression was consistently found in 90% of RPE cells. Within the eye, only the RPE cells were immunoreactive with an anti-cre antibody. Maximum cre expression quantified by Western blot analysis occurred at P28. Crosses with three lines containing floxed sequences revealed RPE-specific cre activity in the eye and extraocular expression limited to the testes. Histology and electroretinography showed no cre-mediated RPE toxicity.
This BEST1-cre transgenic line enables generation of RPE-specific knockout mice. The mosaic expression pattern provides an internal control; the non-cre-expressing RPE cells continue to express the floxed genes. These mice should facilitate study of the multifunctional RPE and the generation of mouse models of human retinal disease.
[Show abstract][Hide abstract] ABSTRACT: Over the last decade, cellular glucose metabolism has emerged as a central player in the mechanisms of programmed cell death (PCD). We examined the metabolic foundations of apoptosis from a Darwinian context and suggest that PCD has evolved from the cellular response to metabolic stress, most notably in relation to glucose metabolism. Whilst apoptosis and other forms of PCD are essential to the development, maintenance and survival of multicellular organisms, it is now evident that controlled and selective cell death confers fitness advantages in unicellular organisms. All species may thus harbour a fundamental relationship between the availability of basic nutrients and life/death decisions. This evolutionary perspective may inform our understanding of PCD in its many guises.
Current opinion in cell biology 10/2009; 21(6):885-93. · 14.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Throughout the lifetime of an individual, light is focused onto the retina. The resulting photooxidative stress can cause acute or chronic retinal damage. The pathogenesis of age-related macular degeneration (AMD), the leading cause of legal blindness in the developed world, involves oxidative stress and death of the retinal pigment epithelium (RPE) followed by death of the overlying photoreceptors. Evidence suggests that damage due to exposure to light plays a role in AMD and other age-related eye diseases. In this work a system for light-induced damage and death of the RPE, based on the human ARPE-19 cell line, was used. Induction of mitochondria-derived reactive oxygen species (ROS) is shown to play a critical role in the death of cells exposed to short-wavelength blue light (425 +/- 20 nm). ROS and cell death are blocked either by inhibiting the mitochondrial electron transport chain or by mitochondria-specific antioxidants. These results show that mitochondria are an important source of toxic oxygen radicals in blue light-exposed RPE cells and may indicate new approaches for treating AMD using mitochondria-targeted antioxidants.
Photochemistry and Photobiology 06/2004; 79(5):470-5. · 2.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To identify and characterize retinoblastoma protein (pRb) binding proteins that may influence retinoblast proliferation and retinal pigment epithelial cell survival.
The yeast two-hybrid system was used to screen a bovine retinal cDNA library and to characterize positive clones. DNA sequencing and site-directed mutagenesis were used for further analysis. Co-immunoprecipitation experiments were used to confirm the results of the two-hybrid system in vivo.
In the two-hybrid system, Protein Phosphatase 1alpha1 (PP1alpha1) binds the retinoblastoma protein. Unlike several other pRb binding proteins, PP1alpha1 binds only weakly to the Rb family member p107, and does not demonstrate detectable binding to p130. Confirming the two-hybrid results, endogenous PP1 in a human retinal pigment epithelial (RPE) cell line co-immunoprecipitates with endogenous pRb but not p107 or p130. Site directed mutagenesis of two pRb binding motifs in PP1alpha1 from LXSXE to LXCXE leads to slight increases in its two-hybrid interaction with pRb but does not alter its binding preference for pRb over the other family members. The complete sequence of bovine PP1alpha1 is reported.
The strong two-hybrid interaction between PP1alpha1 and pRb, but not p107 or p130, suggests that the phosphorylation status of members of the pRb family may be regulated by different phosphatases, contributing to fine control of cell cycle progression. Conversely, PP1 activity may be specifically regulated by pRb and not p107 or p130. Mutagenesis studies suggest that PP1alpha1's LXSXE motif is not responsible for its binding preference for pRb over p107 and p130. Disruption of the PP1-pRb interaction may influence retinoblastoma tumorigenesis as well as RPE cell proliferation and survival.
Current Eye Research 06/2002; 24(5):392-6. · 1.71 Impact Factor