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Edward N Pugh

Publications and source records attributed to Edward N Pugh.

At least 19 recordsLinked to original sources
PubMed2006

Light-driven translocation of signaling proteins in vertebrate photoreceptors.

The dynamic localization of proteins within cells is often determined by environmental stimuli. In retinal photoreceptors, light exposure results in the massive translocation of three key signal transduction proteins, transducin, arrestin and recoverin, into and out of the outer segment compartment where phototransduction takes place. This phenomenon has rapidly taken the center stage of photoreceptor cell biology, thanks to the introduction of new quantitative and transgenic approaches. Here, we discuss evidence that intracellular protein translocation contributes to adaptation of photoreceptors to diurnal changes in ambient light intensity and summarize the current debate on whether it is driven by diffusion or molecular motors.

Animals↗
PubMed2006

RGS expression level precisely regulates the duration of rod photoresponses.

Regulators of G protein signaling (RGS) constitute a family of proteins that bind specifically to the activated alpha subunits of G proteins (Galpha-GTP), acting as GTPase activating proteins, or GAPs, for the rate of GTP hydrolysis. In this issue of Neuron, Krispel et al. resolve a long-standing puzzle in phototransduction, establishing that RGS9 "GAPping" of G(t)alpha-GTP is the molecular mechanism underlying the dominant recovery time constant of mouse rod photoreceptors and that a precise level of expression of RGS9 is required for normal photoresponse timing.

Animals↗
PubMed2006

Adaptive algorithms for two-channel polarization sensing under various polarization statistics with nonuniform distributions.

The polarization of light carries much useful information about the environment. Biological studies have shown that some animal species use polarization information for navigation and other purposes. It has been previously shown that a bioinspired polarization-difference imaging (PDI) technique can facilitate detection and feature extraction of targets in scattering media. It has also been established [J. Opt. Soc. Am. A 15, 359 (1998)] that polarization sum and polarization difference are the optimum pair of linear combinations of images taken through two orthogonally oriented linear polarizers of a scene having a uniform distribution of polarization directions. However, in many real environments the scene has a nonuniform distribution of polarization directions. Using principal component analysis of the polarization statistics of the scene, we develop a method to determine the two optimum information channels with unequal weighting coefficients that can be formed as linear combinations of the images of a scene taken through a pair of linear polarizers not constrained to the horizontal and vertical directions of the scene. We determine the optimal orientations of linear polarization filters that enhance separation of a target from the background, where the target is defined as an area with distinct polarization characteristics as compared to the background. Experimental results confirm that in most situations adaptive PDI outperforms conventional PDI with fixed channels.

Journal Article↗
PubMed2006

Physiological features of the S- and M-cone photoreceptors of wild-type mice from single-cell recordings.

Cone cells constitute only 3% of the photoreceptors of the wild-type (WT) mouse. While mouse rods have been thoroughly investigated with suction pipette recordings of their outer segment membrane currents, to date no recordings from WT cones have been published, likely because of the rarity of cones and the fragility of their outer segments. Recently, we characterized the photoreceptors of Nrl(-/-) mice, using suction pipette recordings from their "inner segments" (perinuclear region), and found them to be cones. Here we report the use of this same method to record for the first time the responses of single cones of WT mice, and of mice lacking the alpha-subunit of the G-protein transducin (G(t)alpha(-/-)), a loss that renders them functionally rodless. Most cones were found to functionally co-express both S- (lambda(max) = 360 nm) and M- (lambda(max) = 508 nm) cone opsins and to be maximally sensitive at 360 nm ("S-cones"); nonetheless, all cones from the dorsal retina were found to be maximally sensitive at 508 nm ("M-cones"). The dim-flash response kinetics and absolute sensitivity of S- and M-cones were very similar and not dependent on which of the coexpressed cone opsins drove transduction; the time to peak of the dim-flash response was approximately 70 ms, and approximately 0.2% of the circulating current was suppressed per photoisomerization. Amplification in WT cones (A approximately 4 s(-2)) was found to be about twofold lower than in rods (A approximately 8 s(-2)). Mouse M-cones maintained their circulating current at very nearly the dark adapted level even when >90% of their M-opsin was bleached. S-cones were less tolerant to bleached S-opsin than M-cones to bleached M-opsin, but still far more tolerant than mouse rods to bleached rhodopsin, which exhibit persistent suppression of nearly 50% of their circulating current following a 20% bleach. Thus, the three types of mouse opsin appear distinctive in the degree to which their bleached, unregenerated opsins generate "dark light."

Animals↗
PubMed2006

Polarization-based and specular-reflection-based noncontact latent fingerprint imaging and lifting.

In forensic science the finger marks left unintentionally by people at a crime scene are referred to as latent fingerprints. Most existing techniques to detect and lift latent fingerprints require application of a certain material directly onto the exhibit. The chemical and physical processing applied to the fingerprint potentially degrades or prevents further forensic testing on the same evidence sample. Many existing methods also have deleterious side effects. We introduce a method to detect and extract latent fingerprint images without applying any powder or chemicals on the object. Our method is based on the optical phenomena of polarization and specular reflection together with the physiology of fingerprint formation. The recovered image quality is comparable to existing methods. In some cases, such as the sticky side of tape, our method shows unique advantages.

Biometry↗
PubMed2005

Mole quantity of RPE65 and its productivity in the generation of 11-cis-retinal from retinyl esters in the living mouse eye.

RPE65, a protein expressed in cells of the retinal pigment epithelium of the eye, is essential for the synthesis by isomerohydrolase of 11-cis-retinal, the chromophore of rod and cone opsins. Recent work has established that RPE65 is a retinyl ester binding protein, and as all-trans-retinyl esters are the substrate for isomerohydrolase activity, the hypothesis has emerged that RPE65 serves to deliver substrate to this enzyme or complex. We bred mice with five distinct combinations of the RPE65 Leu450/Met450 variants (Leu/Leu, Met/Met, Leu/Met, Leu/-, and Met/-), measured in mice of each genotype the mole quantity of RPE65 per eye, and measured the initial rate of rhodopsin regeneration after a nearly complete bleach of rhodopsin to estimate the maximum rate of 11-cis-retinal synthesis in vivo. The quantity of RPE65 per eye ranged from 5.7 pmol (Balb/c) to 0.32 pmol (C57BL/6N x Rpe65(-)(/)(-)); the initial rate of rhodopsin regeneration was a Michaelis function of RPE65, where V(max) = 18 pmol/min per eye and K(m) = 1.7 pmol, and not dependent on the Leu450/Met450 variant. At RPE65 levels well below the K(m), the rate of production of 11-cis-retinal per RPE65 molecule was approximately 10 min(-)(1). Thus, the results imply that as a chaperone each RPE65 molecule can deliver retinyl ester to the isomerohydrolase at a rate of 10 molecules/min; should RPE65 itself be identified as the isomerase, each copy must be able to produce at least 10 molecules of 11-cis-retinal per minute.

Animals↗
PubMed2005

The retinal G protein-coupled receptor (RGR) enhances isomerohydrolase activity independent of light.

Rod and cone visual pigments use 11-cis-retinal, a vitamin A derivative, as their chromophore. Light isomerizes 11-cis- into all-trans-retinal, triggering a conformational transition of the opsin molecule that initiates phototransduction. After bleaching all-trans-retinal leaves the opsin, and light sensitivity must be restored by regeneration of 11-cis-retinal. Under bright light conditions the retinal G protein-coupled receptor (RGR) was reported to support this regeneration by acting as a photoisomerase in a proposed photic visual cycle. We analyzed the contribution of RGR to rhodopsin regeneration under different light regimes and show that regeneration, during light exposure and in darkness, is slowed about 3-fold in Rgr(-/-) mice. These findings are not in line with the proposed function of RGR as a photoisomerase. Instead, RGR, independent of light, accelerates the conversion of retinyl esters to 11-cis-retinal by positively modulating isomerohydrolase activity, a key step in the "classical" visual cycle. Furthermore, we find that light accelerates rhodopsin regeneration, independent of RGR.

Animals↗
PubMed2005

Photoreceptors of Nrl -/- mice coexpress functional S- and M-cone opsins having distinct inactivation mechanisms.

The retinas of mice null for the neural retina leucine zipper transcription factor (Nrl-/-) contain no rods but are populated instead with photoreceptors that on ultrastructural, histochemical, and molecular criteria appear cone like. To characterize these photoreceptors functionally, responses of single photoreceptors of Nrl-/- mice were recorded with suction pipettes at 35-37 degrees C and compared with the responses of rods of WT mice. Recordings were made either in the conventional manner, with the outer segment (OS) drawn into the pipette ("OS in"), or in a novel configuration with a portion of the inner segment drawn in ("OS out"). Nrl-/- photoreceptor responses recorded in the OS-out configuration were much faster than those of WT rods: for dim-flash responses tpeak = 91 ms vs. 215 ms; for saturating flashes, dominant recovery time constants, tau(D) = 110 ms vs. 240 ms, respectively. Nrl-/- photoreceptors in the OS-in configuration had reduced amplification, sensitivity, and slowed recovery kinetics, but the recording configuration had no effect on rod response properties, suggesting Nrl-/- outer segments to be more susceptible to damage. Functional coexpression of two cone pigments in a single mammalian photoreceptor was established for the first time; the responses of every Nrl-/- cell were driven by both the short-wave (S, lambda(max) approximately 360 nm) and the mid-wave (M, lambda(max) approximately 510 nm) mouse cone pigment; the apparent ratio of coexpressed M-pigment varied from 1:1 to 1:3,000 in a manner reflecting a dorso-ventral retinal position gradient. The role of the G-protein receptor kinase Grk1 in cone pigment inactivation was investigated in recordings from Nrl-/-/Grk1-/- photoreceptors. Dim-flash responses of cells driven by either the S- or the M-cone pigment were slowed 2.8-fold and 7.5-fold, respectively, in the absence of Grk1; the inactivation of the M-pigment response was much more seriously retarded. Thus, Grk1 is essential to normal inactivation of both S- and M-mouse cone opsins, but S-opsin has access to a relatively effective, Grk1-independent inactivation pathway.

Animals↗
PubMed2005

Cone-like morphological, molecular, and electrophysiological features of the photoreceptors of the Nrl knockout mouse.

PURPOSE: To test the hypothesis that Nrl(-)(/)(-) photoreceptors are cones, by comparing them with WT rods and cones using morphological, molecular, histochemical, and electrophysiological criteria. METHODS: The photoreceptor layer of fixed retinal tissue of 4- to 6-week-old mice was examined in plastic sections by electron microscopy, and by confocal microscopy in frozen sections immunolabeled for the mouse UV-cone pigment and colabeled with PNA. Quantitative immunoblot analysis was used to determine the levels of expression of key cone-specific proteins. Single- and paired-flash methods were used to extract the spectral sensitivity, kinetics, and amplification of the a-wave of the ERG. RESULTS: Outer segments of Nrl(-/-) photoreceptors ( approximately 7 mum) are shorter than those of wild-type (WT) rods ( approximately 25 mum) and cones ( approximately 15 mum); but, like WT cones, they have 25 or more basal discs open to the extracellular space, extracellular matrix sheaths stained by PNA, chromatin "clumping" in their nuclei, and mitochondria two times shorter than rods. Nrl(-/-) photoreceptors express the mouse UV cone pigment, cone transducin, and cone arrestin in amounts expected, given the relative size and density of cones in the two retinas. The ERG a-wave was used to assay the properties of the photocurrent response. The sensitivity of the Nrl(-/-) a-wave is at its maximum at 360 nm, with a secondary mode at 510 nm having approximately one-tenth the maximum sensitivity. These wavelengths are the lambda(max) of the two mouse cone pigments. The time to peak of the dim-flash photocurrent response was approximately 50 ms, more than two times faster than that of rods. CONCLUSIONS: Many morphological, molecular, and electrophysiological features of the Nrl(-/-) photoreceptors are cone-like, and strongly distinguish these cells from rods. This retina provides a model for the investigation of cone function and cone-specific genetic disease.

Animals↗
PubMed2004

Quantification of the cytoplasmic spaces of living cells with EGFP reveals arrestin-EGFP to be in disequilibrium in dark adapted rod photoreceptors.

The hypothesis is tested that enhanced green fluorescent protein (EGFP) can be used to quantify the aqueous spaces of living cells, using as a model transgenic Xenopus rods. Consistent with the hypothesis, regions of rods having structures that exclude EGFP, such as the mitochondrial-rich ellipsoid and the outer segments, have highly reduced EGFP fluorescence. Over a 300-fold range of expression the average EGFP concentration in the outer segment was approximately half that in the most intensely fluorescent regions of the inner segment, in quantitative agreement with prior X-ray diffraction estimates of outer segment cytoplasmic volume. In contrast, the fluorescence of soluble arrestin-EGFP fusion protein in the dark adapted rod outer segment was approximately threefold lower than predicted by the EGFP distribution, establishing that the fusion protein is not equilibrated with the cytoplasm. Arrestin-EGFP mass was conserved during a large-scale, light-driven redistribution in which approximately 40% of the protein in the inner segment moved to the outer segment in less than 30 minutes.

Animals↗
PubMed2004

From candelas to photoisomerizations in the mouse eye by rhodopsin bleaching in situ and the light-rearing dependence of the major components of the mouse ERG.

To quantify the rate at which light in a ganzfeld produces photoisomerizations in mouse rods in situ, we measured the rate of rhodopsin bleaching in eyes of recently euthanized mice with fully dilated pupils. The amount of rhodopsin declined as a first-order (exponential) function of the duration of the exposure at the luminance of 920 scot cd m(-2): the rate constants of bleaching were 8.3 x 10(-6) and 2.8 x 10(-5) s(-1) (scot cd(-1)m2)(-1) for C57B1/6 and 129P3/J mice, respectively. When the approximately 3-fold difference in effective areas of the pupils of the mice are taken into consideration, the bleaching rates for both strains become essentially the same, 2.6 x 10(-6) fraction rhodopsin (scot Td s)(-1). Assuming 7 x 10(7) rhodopsin molecules per rod, this bleaching rate yields the result that a flash of 1 scot Td s produces 181 photoisomerizations per rod, a value close to that derived from analysis of the collecting area of the rod for axially propagating light. We measured the electroretinograms of mice of the two strains reared under controlled illumination conditions (2 and 100 lux), and compared their properties, using the calibrations to determine the absolute sensitivities of the b-wave and a-waves. The intensity that produces a half-saturating rod b-wave response is 0.3-0.6 photoisomerizations rod(-1), and the amplification constant of the rod a-wave is 5-6 s(-2) photoisomerization(-1), with little dependence on the strain.

Albinism, Ocular↗
PubMed2004

In utero gene therapy rescues vision in a murine model of congenital blindness.

The congenital retinal blindness known as Leber congenital amaurosis (LCA) can be caused by mutations in the RPE65 gene. RPE65 plays a critical role in the visual cycle that produces the photosensitive pigment rhodopsin. Recent evidence from human studies of LCA indicates that earlier rather than later intervention may be more likely to restore vision. We determined the impact of in utero delivery of the human RPE65 cDNA to retinal pigment epithelium cells in a murine model of LCA, the Rpe65(-/-) mouse, using a serotype 2 adeno-associated virus packaged within an AAV1 capsid (AAV2/1). Delivery of AAV2/1-CMV-hRPE65 to fetuses (embryonic day 14) resulted in efficient transduction of retinal pigment epithelium, restoration of visual function, and measurable rhodopsin. The results demonstrate AAV-mediated correction of the deficit and suggest that in utero retinal gene delivery may be a useful approach for treating a variety of blinding congenital retinal diseases.

Animals↗
PubMed2003

Delivery of adeno-associated virus vectors to the fetal retina: impact of viral capsid proteins on retinal neuronal progenitor transduction.

The development of fetal ocular gene transfer may be useful as a therapeutic tool for the prevention of retinal genetic disorders with congenital or early clinical manifestations. In this study we explored the neural progenitor transduction patterns of adeno-associated virus (AAV) vectors following delivery to the developing retina. Recombinant vectors with the same genome carrying the enhanced green fluorescent protein (EGFP) transgene packaged in capsids of differing serotypes (serotypes 1, 2, and 5, termed AAV2/1, AAV2/2, and AAV2/5, respectively) were created. Delivery of the AAV vectors during early retinal development resulted in efficient and stable transduction of retinal progenitors. Vector surface proteins and the developmental status of the retina profoundly affected viral tropism and transgene distribution. The procedure is not detrimental to retinal development and function and therefore provides a safe delivery vehicle for potential therapeutic applications and a means of assessing the mechanisms of retina development and disease.

Animals↗
PubMed2003

RP1 is required for the correct stacking of outer segment discs.

PURPOSE: Mutations in RP1 are a common cause of dominant retinitis pigmentosa (RP), but the mechanism by which the identified mutations lead to photoreceptor cell death and blindness has not been determined. To investigate the function of the RP1 protein in photoreceptors and gain insight into the mechanism of disease, gene-targeting techniques were used to produce mice with a mutant Rp1 allele that mimics the truncation alleles found to cause disease. METHODS: RT-PCR was used to amplify illegitimate RP1 transcripts from lymphoblasts. Gene targeting was used to create mice with a mutant Rp1-myc allele. Confocal immunofluorescence microscopy was used to identify the location of the mutant Rp1-myc protein in photoreceptors. The structure of the photoreceptors in the resultant Rp1-myc mice was studied by light and electron microscopy. The retinal function of the mutant mice was investigated using analysis of full-field ERGs. RESULTS: Wild-type and mutant RP1 mRNA were both detected in lymphoblasts from patients with RP1 disease. Rp1-myc mice produced a truncated version of the Rp1 protein, containing the N-terminal 662 amino acids, which localized correctly to the axoneme of the photoreceptor outer segments. Mice homozygous for the mutant Rp1-myc allele underwent a rapid-onset retinal degeneration characterized by incorrectly oriented outer segment discs that failed to stack properly into outer segments. In contrast, the photoreceptors of heterozygous mice remained relatively healthy. CONCLUSIONS: The presence of mutant RP1 mRNA in lymphoblasts from patients with RP1 disease implies that the mutant message can escape nonsense-mediated mRNA decay and that a truncated RP1 protein may be produced in the retina. The truncated Rp1-myc protein appears to be nonfunctional, and not to exert a dominant negative effect in the photoreceptors of heterozygous mice. Results from homozygous Rp1-myc mice indicate that RP1 is required for the correct orientation and higher order stacking of outer segment discs.

Animals↗
PubMed2002

Connexin36 forms synapses essential for night vision.

Rod signals reach ganglion cells via at least five pathways. A new study in this issue of Neuron shows that all pathways from rods to ON-type ganglion cells require connexin36. This is the first physiological demonstration in mammals that a well-defined neural circuit requires a gap junction as an obligatory transmission step.

Animals↗
PubMed2002

Massive light-driven translocation of transducin between the two major compartments of rod cells: a novel mechanism of light adaptation.

We report a new cellular mechanism of rod photoreceptor adaptation in vivo, which is triggered by daylight levels of illumination. The mechanism involves a massive light-dependent translocation of the photoreceptor-specific G protein, transducin, between the functional compartments of rods. To characterize the mechanism, we developed a novel technique that combines serial tangential cryodissection of the rat retina with Western blot analysis of protein distribution in the sections. Up to 90% of transducin translocates from rod outer segments to other cellular compartments on the time scale of tens of minutes. The reduction in the transducin content of the rod outer segments is accompanied by a corresponding reduction in the amplification of the rod photoresponse, allowing rods to operate in illumination up to 10-fold higher than would otherwise be possible.

Adaptation, Ocular↗