6 research outputs found
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A nonhuman primate model of inherited retinal disease.
Inherited retinal degenerations are a common cause of untreatable blindness worldwide, with retinitis pigmentosa and cone dystrophy affecting approximately 1 in 3500 and 1 in 10,000 individuals, respectively. A major limitation to the development of effective therapies is the lack of availability of animal models that fully replicate the human condition. Particularly for cone disorders, rodent, canine, and feline models with no true macula have substantive limitations. By contrast, the cone-rich macula of a nonhuman primate (NHP) closely mirrors that of the human retina. Consequently, well-defined NHP models of heritable retinal diseases, particularly cone disorders that are predictive of human conditions, are necessary to more efficiently advance new therapies for patients. We have identified 4 related NHPs at the California National Primate Research Center with visual impairment and findings from clinical ophthalmic examination, advanced retinal imaging, and electrophysiology consistent with achromatopsia. Genetic sequencing confirmed a homozygous R565Q missense mutation in the catalytic domain of PDE6C, a cone-specific phototransduction enzyme associated with achromatopsia in humans. Biochemical studies demonstrate that the mutant mRNA is translated into a stable protein that displays normal cellular localization but is unable to hydrolyze cyclic GMP (cGMP). This NHP model of a cone disorder will not only serve as a therapeutic testing ground for achromatopsia gene replacement, but also for optimization of gene editing in the macula and of cone cell replacement in general
Comparative Analysis of Cone and Rod Transducins Using Chimeric Gα Subunits
The molecular nature of transducin-α subunits (Gα<sub>t</sub>) may contribute to the distinct physiology of cone and rod
photoreceptors. Biochemical properties of mammalian cone Gα<sub>t2</sub> subunits and their differences with rod Gα<sub>t1</sub> are largely unknown. Here, we examined properties of chimeric Gα<sub>t2</sub> in comparison with its rod counterpart. The key biochemical
difference between the rod- and cone-like Gα<sub>t</sub> was
∼10-fold higher intrinsic nucleotide exchange on the chimeric
Gα<sub>t2</sub>. Presented mutational analysis suggests that
weaker interdomain interactions between the GTPase (Ras-like) domain
and the helical domain in Gα<sub>t2</sub> are in part responsible
for its increased spontaneous nucleotide exchange. However, the rates
of R*-dependent nucleotide exchange of chimeric Gα<sub>t2</sub> and Gα<sub>t1</sub> were equivalent. Furthermore, chimeric
Gα<sub>t2</sub> and Gα<sub>t1</sub> exhibited similar
rates of intrinsic GTPase activity as well as similar acceleration
of GTP hydrolysis by the RGS domain of RGS9. Our results suggest that
the activation and inactivation properties of cone and rod Gα<sub>t</sub> subunits in an <i>in vitro</i> reconstituted system
are comparable
Identification and Characterization of a Mandelamide Hydrolase and an NAD(P)(+)-Dependent Benzaldehyde Dehydrogenase from Pseudomonas putida ATCC 12633
The enzymes of the mandelate metabolic pathway permit Pseudomonas putida ATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarboxylase arranged in an operon. Here we report the sequencing of the remainder of the restriction fragment, which revealed three further open reading frames, denoted mdlX, mdlY, and mdlD. All were transcribed in the opposite direction from the genes of the mdlABC operon. Sequence alignments suggested that the open reading frames encoded a regulatory protein (mdlX), a member of the amidase signature family (mdlY), and an NAD(P)(+)-dependent dehydrogenase (mdlD). The mdlY and mdlD genes were isolated and expressed in Escherichia coli, and the purified gene products were characterized as a mandelamide hydrolase and an NAD(P)(+)-dependent benzaldehyde dehydrogenase, respectively
Recommended from our members
A nonhuman primate model of inherited retinal disease.
Inherited retinal degenerations are a common cause of untreatable blindness worldwide, with retinitis pigmentosa and cone dystrophy affecting approximately 1 in 3500 and 1 in 10,000 individuals, respectively. A major limitation to the development of effective therapies is the lack of availability of animal models that fully replicate the human condition. Particularly for cone disorders, rodent, canine, and feline models with no true macula have substantive limitations. By contrast, the cone-rich macula of a nonhuman primate (NHP) closely mirrors that of the human retina. Consequently, well-defined NHP models of heritable retinal diseases, particularly cone disorders that are predictive of human conditions, are necessary to more efficiently advance new therapies for patients. We have identified 4 related NHPs at the California National Primate Research Center with visual impairment and findings from clinical ophthalmic examination, advanced retinal imaging, and electrophysiology consistent with achromatopsia. Genetic sequencing confirmed a homozygous R565Q missense mutation in the catalytic domain of PDE6C, a cone-specific phototransduction enzyme associated with achromatopsia in humans. Biochemical studies demonstrate that the mutant mRNA is translated into a stable protein that displays normal cellular localization but is unable to hydrolyze cyclic GMP (cGMP). This NHP model of a cone disorder will not only serve as a therapeutic testing ground for achromatopsia gene replacement, but also for optimization of gene editing in the macula and of cone cell replacement in general