Glutamine is the only amino acid that can adequately support the generation of NADPH in rod photoreceptors.

NADPH, the primary source of reducing equivalents in the cytosol, is used in vertebrate rod photoreceptor outer segments to reduce the all-trans retinal released from photoactivated visual pigment to all-trans retinol. Light activation of the visual pigment isomerizes the 11-cis retinal chromophore to all-trans, thereby destroying it and necessitating its regeneration. Release and reduction of all-trans retinal are the first steps in the series of reactions that regenerate the visual pigment. Glucose and glutamine can both support the reduction of all-trans retinal to retinol, indicating that the NADPH used in rod photoreceptor outer segments can be generated by the pentose phosphate pathway as well as by mitochondria-linked pathways. We have used the conversion of all-trans retinal to all-trans retinol to examine whether amino acids other than glutamine can also support the generation of NADPH in rod photoreceptors. We have measured this conversion in single isolated mouse rod photoreceptors by imaging the fluorescence of the all-trans retinal and retinol generated after exposure of the cells to light. In agreement with previous work, we find that 5 mM glucose or 0.5 mM glutamine support the conversion of ∼70-80% of all-trans retinal to retinol, corresponding to a reduced NADP fraction of ∼10%. All other amino acids at 0.5 mM concentration support the conversion to a much lesser extent, indicating reduced NADP fractions of 1-2% at most. Taurine was also ineffective at supporting NADPH generation, while formic acid, the toxic metabolite of methanol, suppressed the generation of NADPH by either glucose or glutamine.

Evidence for ceramide induced cytotoxicity in retinal ganglion cells.

Ceramides are bioactive compounds that play important roles in regulating cellular responses to extracellular stimuli and stress. Previous studies have shown that ceramides contribute to retinal degeneration associated with ischemic and ocular hypertensive stress. Acid sphingomyelinase (ASMase) is one of the major enzymes responsible for the stress-induced generation of ceramides. The goals of this study are to investigate the effects of ceramides on retinal ganglion cells (RGCs) and of ASMase inhibition in ocular hypertensive mice. Induced pluripotent stem cell (iPSC)-derived RGCs and primary cultures of human optic nerve head astrocytes were used to characterize the response to C2-ceramide. Microbead-induced ocular hypertension in the ASMase heterozygote mouse model was used to confirm the physiological relevance of in vitro studies. In mice, RGC function and morphology were assessed with pattern ERG (pERG) and immunofluorescence. The addition of C2-ceramide to iPSC-derived RGCs produced a significant concentration- and time-dependent reduction in cell numbers when compared to control cultures. While the addition of C2-ceramide to astrocytes did not affect viability, it resulted in a 2.6-fold increase in TNF-α secretion. The addition of TNF-α or conditioned media from C2-ceramide-treated astrocytes to RGC cultures significantly reduced cell numbers by 56.1 ± 8.4% and 24.7 ± 4.8%, respectively. This cytotoxic response to astrocyte-conditioned media was blocked by TNF-α antibody. In ASMase heterozygote mice, functional and morphological analyses of ocular hypertensive eyes reveal significantly less RGC degeneration when compared with hypertensive eyes from wild-type mice. These results provide evidence that ceramides can induce RGC cell death by acting directly, as well as indirectly via the secretion of TNF-α from optic nerve head astrocytes. In vivo studies in mice provide evidence that ceramides derived through the activity of ASMase contribute to ocular hypertensive injury. Together these results support the importance of ceramides in the pathogenesis of ocular hypertensive injury to the retina.

Photooxidation mediated by 11-cis and all-trans retinal in single isolated mouse rod photoreceptors.

Retinal, the vitamin A aldehyde, is a potent photosensitizer that plays a major role in light-induced damage to vertebrate photoreceptors. 11-Cis retinal is the light-sensitive chromophore of rhodopsin, the photopigment of vertebrate rod photoreceptors. It is isomerized by light to all-trans, activating rhodopsin and beginning the process of light detection. All-trans retinal is released by activated rhodopsin, allowing its regeneration by fresh 11-cis retinal continually supplied to photoreceptors. The released all-trans retinal is reduced to all-trans retinol in a reaction using NADPH. We have examined the photooxidation mediated by 11-cis and all-trans retinal in single living rod photoreceptors isolated from mouse retinas. Photooxidation was measured with fluorescence imaging from the oxidation of internalized BODIPY C11, a fluorescent dye whose fluorescence changes upon oxidation. We found that photooxidation increased with the concentration of exogenously added 11-cis or all-trans retinal to metabolically compromised rod outer segments that lacked NADPH supply. In dark-adapted metabolically intact rod outer segments with access to NADPH, there was no significant increase in photooxidation following exposure of the cell to light, but there was significant increase following addition of exogenous 11-cis retinal. The results indicate that both 11-cis and all-trans retinal can mediate light-induced damage in rod photoreceptors. In metabolically intact cells, the removal of the all-trans retinal generated by light through its reduction to retinol minimizes all-trans retinal-mediated photooxidation. However, because the enzymatic machinery of the rod outer segment cannot remove 11-cis retinal, 11-cis-retinal-mediated photooxidation may play a significant role in light-induced damage to photoreceptor cells.

Interphotoreceptor retinoid-binding protein removes all-trans-retinol and retinal from rod outer segments, preventing lipofuscin precursor formation.

Interphotoreceptor retinoid-binding protein (IRBP) is a specialized lipophilic carrier that binds the all-trans and 11-cis isomers of retinal and retinol, and this facilitates their transport between photoreceptors and cells in the retina. One of these retinoids, all-trans-retinal, is released in the rod outer segment by photoactivated rhodopsin after light excitation. Following its release, all-trans-retinal is reduced by the retinol dehydrogenase RDH8 to all-trans-retinol in an NADPH-dependent reaction. However, all-trans-retinal can also react with outer segment components, sometimes forming lipofuscin precursors, which after conversion to lipofuscin accumulate in the lysosomes of the retinal pigment epithelium and display cytotoxic effects. Here, we have imaged the fluorescence of all-trans-retinol, all-trans-retinal, and lipofuscin precursors in real time in single isolated mouse rod photoreceptors. We found that IRBP removes all-trans-retinol from individual rod photoreceptors in a concentration-dependent manner. The rate constant for retinol removal increased linearly with IRBP concentration with a slope of 0.012 min-1 µm-1 IRBP also removed all-trans-retinal, but with much less efficacy, indicating that the reduction of retinal to retinol promotes faster clearance of the photoisomerized rhodopsin chromophore. The presence of physiological IRBP concentrations in the extracellular medium resulted in lower levels of all-trans-retinal and retinol in rod outer segments following light exposure. It also prevented light-induced lipofuscin precursor formation, but it did not remove precursors that were already present. These findings reveal an important and previously unappreciated role of IRBP in protecting the photoreceptor cells against the cytotoxic effects of accumulated all-trans-retinal.

Hepatic stellate cells retain retinoid-laden lipid droplets after cellular transdifferentiation into activated myofibroblasts.

Loss of retinyl ester (RE)-rich lipid droplets (LDs) from hepatic stellate cells (HSCs) is cited as a key event in their cellular transdifferentiation to activated, pro-fibrotic myofibroblasts; however, it remains unclear if changes in LD morphology or RE content are causal for transdifferentiation. To better understand LD dynamics in vitro within a common model of HSC activation, we used novel approaches preserving LD morphology and allowing for quantitation of RE. The size and quantity of LDs within in vitro and in vivo bile duct ligation (BDL)-activated HSCs were quantitated using adipocyte differentiation-related protein (ADRP) labeling and oil red o (ORO) staining (gold standard), and RE content was determined using fluorescence microscopy. We found during HSC activation in vitro that LD number differed significantly when measured by ADRP and ORO, respectively ( day 1: 56 vs. 5, P = 0.03; day 4: 101 vs. 39, P = 0.03; day 14: 241 vs. 12, P = 0.02). Ex vivo HSCs activated in vivo contained the same number of LDs as day 4 in vitro activated HSCs (118 vs. 101, P = 0.54). Decline in LD RE occurred beyond day 4 in vitro and day 1 ex vivo , after HSC transdifferentiation was underway. Lastly, in situ HSCs examined using electron microscopy show LDs tend to be smaller but are ultimately retained in BDL injured livers. Therefore, we conclude that during HSC transdifferentiation, LDs are not lost but are retained, decreasing in size. Additionally, RE content declines after transdifferentiation is underway. These data suggest that these LD changes are not causal for HSC transdifferentiation. NEW & NOTEWORTHY Loss of retinoid-laden lipid droplets from hepatic stellate cells has long been held as a hallmark of their transdifferentiation into activated myofibroblasts, the dominant cells that drive hepatic fibrosis. This study demonstrates that stellate cells activated in culture and after liver injury in vivo retain their lipid droplets and that these droplets become smaller and more numerous, with decreases in droplet retinoid concentration occurring only after cellular transdifferentiation is underway.

All-trans retinal levels and formation of lipofuscin precursors after bleaching in rod photoreceptors from wild type and Abca4-/- mice.

The accumulation of lipofuscin in the cells of the retinal pigment epithelium (RPE) is thought to play an important role in the development and progression of degenerative diseases of the retina. The bulk of RPE lipofuscin originates in reactions of the rhodopsin chromophore, retinal, with components of the photoreceptor outer segment. The 11-cis retinal isomer is generated in the RPE and supplied to rod photoreceptor outer segments where it is incorporated as the chromophore of rhodopsin. It is photoisomerized during light detection to all-trans and subsequently released by photoactivated rhodopsin as all-trans retinal, which is removed through reduction to all-trans retinol in a reaction requiring metabolic input in the form of NADPH. Both 11-cis and all-trans retinal can form lipofuscin precursor fluorophores in rod photoreceptor outer segments. Increased accumulation of lipofuscin has been suggested to result from excess formation of lipofuscin precursors due to buildup of all-trans retinal released by light exposure. In connection with this suggestion, the Abca4 transporter protein, an outer segment protein defects in which result in recessive Stargardt disease, has been proposed to promote the removal of all-trans retinal by facilitating its availability for reduction. To examine this possibility, we have measured the outer segment levels of all-trans retinal, all-trans retinol, and of lipofuscin precursors after bleaching by imaging the fluorescence of single rod photoreceptors isolated from wild type and Abca4-/- mice. We found that all-trans retinol and all-trans retinal levels increased after bleaching in both wild type and Abca4-/- rods. At all times after bleaching, there was no significant difference in all-trans retinal levels between the two strains. All-trans retinol levels were not significantly different between the two strains at early times, but were lower in Abca4-/- rods at times longer than 20 min after bleaching. Bleaching in the presence of lower metabolic substrate concentrations resulted in higher all-trans retinal levels and increased formation of lipofuscin precursors in both wild type and Abca4-/- rods. The results show that conditions that result in buildup of all-trans retinal levels result in increased generation of lipofuscin precursors in both wild type and Abca4-/- rods. The results are consistent with the proposal that Abca4 facilitates the reduction of all-trans retinal to retinol; absence of Abca4 however does not appear to be associated with higher all-trans retinal levels compared to wild type.

Mitochondria contribute to NADPH generation in mouse rod photoreceptors.

NADPH is the primary source of reducing equivalents in the cytosol. Its major source is considered to be the pentose phosphate pathway, but cytosolic NADP(+)-dependent dehydrogenases using intermediates of mitochondrial pathways for substrates have been known to contribute. Photoreceptors, a nonproliferating cell type, provide a unique model for measuring the functional utilization of NADPH at the single cell level. In these cells, NADPH availability can be monitored from the reduction of the all-trans-retinal generated by light to all-trans-retinol using single cell fluorescence imaging. We have used mouse rod photoreceptors to investigate the generation of NADPH by different metabolic pathways. In the absence of extracellular metabolic substrates, NADPH generation was severely compromised. Extracellular glutamine supported NADPH generation to levels comparable to those of glucose, but pyruvate and lactate were relatively ineffective. At low extracellular substrate concentrations, partial inhibition of ATP synthesis lowered, whereas suppression of ATP consumption augmented NADPH availability. Blocking pyruvate transport into mitochondria decreased NADPH availability, and addition of glutamine restored it. Our findings demonstrate that in a nonproliferating cell type, mitochondria-linked pathways can generate substantial amounts of NADPH and do so even when the pentose phosphate pathway is operational. Competing demands for ATP and NADPH at low metabolic substrate concentrations indicate a vulnerability to nutrient shortages. By supporting substantial NADPH generation, mitochondria provide alternative metabolic pathways that may support cell function and maintain viability under transient nutrient shortages. Such pathways may play an important role in protecting against retinal degeneration.

The First Steps of the Visual Cycle in Human Rod and Cone Photoreceptors.

Purpose: Light detection destroys the visual pigment. Its regeneration, necessary for the recovery of light sensitivity, is accomplished through the visual cycle. Release of all-trans retinal by the light-activated visual pigment and its reduction to all-trans retinol comprise the first steps of the visual cycle. In this study, we determined the kinetics of all-trans retinol formation in human rod and cone photoreceptors. Methods: Single living rod and cone photoreceptors were isolated from the retinas of human cadaver eyes (ages 21 to 90 years). Formation of all-trans retinol was measured by imaging its outer segment fluorescence (excitation, 360 nm; emission, >420 nm). The extent of conversion of released all-trans retinal to all-trans retinol was determined by measuring the fluorescence excited by 340 and 380 nm. Measurements were repeated with photoreceptors isolated from Macaca fascicularis retinas. Experiments were carried out at 37°C. Results: We found that ∼80% to 90% of all-trans retinal released by the light-activated pigment is converted to all-trans retinol, with a rate constant of 0.24 to 0.55 min-1 in human rods and ∼1.8 min-1 in human cones. In M. fascicularis rods and cones, the rate constants were 0.38 ± 0.08 min-1 and 4.0 ± 1.1 min-1, respectively. These kinetics are several times faster than those measured in other vertebrates. Interphotoreceptor retinoid-binding protein facilitated the removal of all-trans retinol from human rods. Conclusions: The first steps of the visual cycle in human photoreceptors are several times faster than in other vertebrates and in line with the rapid recovery of light sensitivity exhibited by the human visual system.

Reduction of all-trans-retinal in vertebrate rod photoreceptors requires the combined action of RDH8 and RDH12.

In vertebrate rod cells, retinoid dehydrogenases/reductases (RDHs) are critical for reducing the reactive aldehyde all-trans-retinal that is released by photoactivated rhodopsin, to all-trans-retinol (vitamin A). Previous studies have shown that RDH8 localizes to photoreceptor outer segments and is a strong candidate for performing this role. However, RDH12 function in the photoreceptor inner segments is also key, because loss of function mutations cause retinal degeneration in some forms of Leber congenital amaurosis. To investigate the in vivo roles of RDH8 and RDH12, we used fluorescence imaging to examine all-trans-retinol production in single isolated rod cells from wild-type mice and knock-out mice lacking either one or both RDHs. Outer segments of rods deficient in Rdh8 failed to reduce all-trans-retinal, but those deficient in Rdh12 were unaffected. Following exposure to light, a leak of retinoids from outer to inner segments was detected in rods from both wild-type and knock-out mice. In cells lacking Rdh8 or Rdh12, this leak was mainly all-trans-retinal. Wild-type rods incubated with all-trans-retinal reduced moderate loads of retinal within the cell interior, but this ability was lost by cells deficient in Rdh8 or Rdh12. Our findings are consistent with localization of RDH8 to the outer segment where it provides most of the activity needed to reduce all-trans-retinal generated by the light response. In contrast, RDH12 in inner segments can protect vital cell organelles against aldehyde toxicity caused by an intracellular leak of all-trans-retinal, as well as other aldehydes originating both inside and outside the cell.

Lipofuscin and N-retinylidene-N-retinylethanolamine (A2E) accumulate in retinal pigment epithelium in absence of light exposure: their origin is 11-cis-retinal.

The age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been associated with the development of retinal diseases, particularly age-related macular degeneration and Stargardt disease. A major component of lipofuscin is the bis-retinoid N-retinylidene-N-retinylethanolamine (A2E). The current model for the formation of A2E requires photoactivation of rhodopsin and subsequent release of all-trans-retinal. To understand the role of light exposure in the accumulation of lipofuscin and A2E, we analyzed RPEs and isolated rod photoreceptors from mice of different ages and strains, reared either in darkness or cyclic light. Lipofuscin levels were determined by fluorescence imaging, whereas A2E levels were quantified by HPLC and UV-visible absorption spectroscopy. The identity of A2E was confirmed by tandem mass spectrometry. Lipofuscin and A2E levels in the RPE increased with age and more so in the Stargardt model Abca4(-/-) than in the wild type strains 129/sv and C57Bl/6. For each strain, the levels of lipofuscin precursor fluorophores in dark-adapted rods and the levels and rates of increase of RPE lipofuscin and A2E were not different between dark-reared and cyclic light-reared animals. Both 11-cis- and all-trans-retinal generated lipofuscin-like fluorophores when added to metabolically compromised rod outer segments; however, it was only 11-cis-retinal that generated such fluorophores when added to metabolically intact rods. The results suggest that lipofuscin originates from the free 11-cis-retinal that is continuously supplied to the rod for rhodopsin regeneration and outer segment renewal. The physiological role of Abca4 may include the translocation of 11-cis-retinal complexes across the disk membrane.

Rapid formation of all-trans retinol after bleaching in frog and mouse rod photoreceptor outer segments.

All-trans retinol is formed in the outer segments of vertebrate rod photoreceptors from the reduction of the all-trans retinal released by photoactivated rhodopsin. The reduction requires NADPH and is therefore dependent on metabolic input. In metabolically intact photoreceptors, a large increase in rod outer segment fluorescence, attributed to the fluorescence of all-trans retinol, follows rhodopsin photoactivation. The fluorescence increase is biphasic, including a rapid and a slow component. In metabolically compromised cells, there is a much smaller fluorescence increase following rhodopsin photoactivation, but it too contains a rapid component. We have measured the fluorescence signal in single living frog and mouse rod photoreceptors, and have characterized its dependence on the wavelengths of light selected for excitation and for collecting emission. We find that in metabolically intact cells, the excitation and emission properties of both the rapid and slow components of the fluorescence signal are in close agreement with those of all-trans retinol fluorescence. In metabolically compromised cells, however, the signal can only partially be due to all-trans retinol, and most of it is consistent with all-trans retinal. The results suggest that in the outer segments of living rod photoreceptors there is rapid release of all-trans retinal, which in metabolically intact cells is accompanied by rapid conversion to all-trans retinol.

Longitudinal diffusion of a polar tracer in the outer segments of rod photoreceptors from different species.

Vertebrate rod photoreceptors are the ultimate light sensors, as they can detect a single photon. In darkness, rods maintain a high concentration of the intracellular messenger cyclic guanosine monophosphate (cGMP), which binds to and keeps open cationic channels on the plasma membrane of the outer segment. Absorption of a photon by the visual pigment of the rod, rhodopsin, initiates a biochemical amplification cascade that leads to a reduction in the concentration of cGMP and closure of the channels, thereby converting the incoming light to an electrical signal. Because the absorption of a photon and the ensuing reactions are localized events, the magnitude of the response of the rod to a single photon depends on the spread of the decrease in the cGMP concentration along the length of the outer segment. The longitudinal diffusion of cGMP depends on the structural parameters of the rod outer segment, specifically the area and the volume available for diffusion. To characterize the effect of rod outer segment cytoarchitecture on diffusion, we have used fluorescence recovery after photobleaching (FRAP) and examined the mobility of a fluorescent polar tracer, calcein, in the rod outer segments from three species with different outer segment structures: frog (Rana pipiens), mouse (Mus musculus domesticus) and gecko (Gekko gekko). We found that the diffusion coefficient is similar for all three species, in the order of 8-17 microm(2) s(-1), in broad agreement with the predictions by Holcman and Korenbrot (Biophys. J. 2004:86;2566-2582) based on the known cytoarchitecture of rod outer segments. Consequently, the results also support their prediction that the longitudinal spread of light excitation in rods is similar across species.

Physiological and microfluorometric studies of reduction and clearance of retinal in bleached rod photoreceptors.

The visual cycle comprises a sequence of reactions that regenerate the visual pigment in photoreceptors during dark adaptation, starting with the reduction of all-trans retinal to all-trans retinol and its clearance from photoreceptors. We have followed the reduction of retinal and clearance of retinol within bleached outer segments of red rods isolated from salamander retina by measuring its intrinsic fluorescence. Following exposure to a bright light (bleach), increasing fluorescence intensity was observed to propagate along the outer segments in a direction from the proximal region adjacent to the inner segment toward the distal tip. Peak retinol fluorescence was achieved after approximately 30 min, after which it declined very slowly. Clearance of retinol fluorescence is considerably accelerated by the presence of the exogenous lipophilic substances IRBP (interphotoreceptor retinoid binding protein) and serum albumin. We have used simultaneous fluorometric and electrophysiological measurements to compare the rate of reduction of all-trans retinal to all-trans retinol to the rate of recovery of flash response amplitude in these cells in the presence and absence of IRBP. We find that flash response recovery in rods is modestly accelerated in the presence of extracellular IRBP. These results suggest such substances may participate in the clearance of retinoids from rod photoreceptors, and that this clearance, at least in rods, may facilitate dark adaptation by accelerating the clearance of photoproducts of bleaching.

Kinetics of rhodopsin's chromophore monitored in a single photoreceptor.

Absorption of light isomerizes the retinyl chromophore of the photoreceptor pigment rhodopsin from 11-cis to all-trans, generating the photoactivated rhodopsin form. The photoisomerization of the chromophore however destroys rhodopsin, and its regeneration requires the removal of the all-trans and the supply of fresh 11-cis chromophore. The all-trans chromophore is removed through a series of steps beginning with its release from photoactivated rhodopsin in the form of all-trans-retinal, leaving behind the apoprotein opsin. All-trans-retinal is then reduced to all-trans-retinol, which is transported out of the photoreceptor. Rhodopsin is regenerated from opsin and fresh 11-cis-retinal arriving to the photoreceptor from the retinal pigment epithelium. Both all-trans and 11-cis-retinal can form precursors of lipofuscin, a pigment that accumulates with age in the lysosomal compartment of the retinal pigment epithelium. All-trans-retinal, all-trans-retinol, and lipofuscin precursors all emit significant and distinct fluorescence signals, allowing their monitoring in single photoreceptor cells with fluorescence imaging. Here we describe the procedures for measuring these fluorophores in single mouse rod photoreceptors.

The 11-cis Retinal Origins of Lipofuscin in the Retina.

Lipofuscin is a fluorescent mixture of partially digested proteins and lipids that accumulates with age in the lysosomal compartment of the retinal pigment epithelium (RPE) of the eye. Because it has been found to have significant cytotoxic potential, lipofuscin is thought to play a role in retinal degeneration diseases including age-related macular degeneration and Stargardt disease, a form of juvenile macular degeneration. The only known components of lipofuscin are bis-retinoids, the condensation products of two molecules of retinal. The bulk of lipofuscin is thought to originate in the rod photoreceptor outer segments as a by-product of reactions involving the retinal chromophore of rhodopsin. 11-cis retinal flows from the RPE into the rod outer segments, where it combines with opsin to form rhodopsin; all-trans retinal is released into the rod outer segments by photoactivated rhodopsin following its excitation by light. Both 11-cis and all-trans retinal can generate lipofuscin-like fluorophores and bis-retinoids when added to rod outer segment membranes. The levels of lipofuscin precursor fluorophores present in the outer segments of dark-adapted rods are similar in cyclic-light- and dark-reared mice, as are the levels of accumulated lipofuscin in the RPE. Because the retinol dehydrogenase enzyme present in rod outer segments can reduce all-trans but not 11-cis retinal, lipofuscin precursors are more likely to form from 11-cis than all-trans retinal, even under cyclic light conditions. Thus, 11-cis retinal may be the primary source of lipofuscin in the retina.

Aldh3a1 protects human corneal epithelial cells from ultraviolet- and 4-hydroxy-2-nonenal-induced oxidative damage.

Aldehyde dehydrogenase 3A1 (ALDH3A1) is one of the most abundant proteins found in corneal epithelial cells of mammalian species, with several postulated protective roles that include detoxification of peroxidic aldehydes, scavenging of free radicals, and direct absorption of ultraviolet (UV) radiation. In the present study, the protective role of ALDH3A1 against UV- and 4-hydroxy-2-nonenal- (4-HNE-) induced oxidative damage was studied. For this purpose, human ALDH3A1 was stably transfected in a human corneal epithelial cell line (HCE) lacking endogenous enzyme. Cells transfected with ALDH3A1 were more resistant to UV- and 4-HNE-induced cytotoxicity than mock-transfected cells. DNA fragmentation assays revealed that both treatments induced apoptosis in mock-transfected cells, but not in ALDH3A1-expressing cells. Apoptosis appeared to occur via caspase-3 activation and subsequent PARP cleavage. The Michaelis-Menten constant (K(m)) for 4-HNE was 54 microM in ALDH3A1-transfected cells; the addition of 100 microM 4-HNE increased NAD(P)H levels by 50% above that in mock-transfected cells. We also found that ALDH3A1 expression prevented 4-HNE-induced protein adduct formation. Taken together, these data suggest that ALDH3A1 is a regulatory element of the cellular defense system that protects corneal epithelium against UV- and 4-HNE-induced oxidative damage.

Calcium and phototransduction.

Visual phototransduction, the conversion of incoming light to an electrical signal, takes place in the outer segments of the rod and cone photoreceptor cells. Light reduces the concentration of cGMP, which, in darkness, keeps open cationic channels present in the plasma membrane of the outer segment. Ca2+ plays an important role in phototransduction by modulating the cGMP-gated channels as well as cGMP synthesis and breakdown. Ca2+ is involved in a negative feedback that is essential for photoreceptor adaptation to background illumination. The effects of Ca2+ on the different components of rod phototransduction have been characterized and can quantitatively account for the steady state responses of the rod cell to background illumination. The propagation of the Ca2+ feedback signal from the periphery toward the center of the outer segment depends on the Ca2+ diffusion coefficient, which has a value of 15 +/- 1 microm2 s(-1). This value shows that diffusion of Ca2+ in the radial direction is quite slow providing a significant barrier in the propagation of the feedback signal. Also, because the diffusion coefficient of Ca2+ is much smaller than that of cGMP, the decline of Ca2+ in the longitudinal direction lags behind the propagation of excitation by the decline of cGMP.

All-trans retinal mediates light-induced oxidation in single living rod photoreceptors.

All-trans retinal is a potent photosensitizer that is released in photoreceptor outer segments by the photoactivated visual pigment following the detection of light. Photoreceptor outer segments also contain high concentrations of polyunsaturated fatty acids, and are thus particularly susceptible to oxidative damage such as that initiated by light via a photosensitizer. Upon its release, all-trans retinal is reduced within the outer segment to all-trans retinol, through a reaction requiring metabolic input in the form of NADPH. The phototoxic potential of physiologically generated all-trans retinal was examined in single living rod photoreceptors obtained from frog (Rana pipiens) retinas. Light-induced oxidation was measured with fluorescence imaging using an oxidation-sensitive indicator dye from the shift in fluorescence between the intact and oxidized forms. Light-induced oxidation was highest in metabolically compromised rod outer segments following photoactivation of the visual pigment rhodopsin, and after a time interval, sufficiently long to ensure the release of all-trans retinal. Furthermore, light-induced oxidation increased with the concentration of exogenously added all-trans retinal. The results show that the all-trans retinal generated during the detection of light can mediate light-induced oxidation. Its removal through reduction to all-trans retinol protects photoreceptor outer segments against light-induced oxidative damage.

[Analysis of schistosomiasis endemic situation in Jiujiang City, 2011].

OBJECTIVE: To understand the endemic situation of schistosomiasis in Jiujiang City in 2011, so as to provide the evidence for decision-making of schistosomiasis control. METHODS: The epidemiological data of schistosomiasis in Jiujiang in 2011 were collected and analyzed with the descriptive epidemiology method. RESULTS: By the end of 2011, there were 34974 cases of schistosomiasis in Jiujiang City, among them there were 2 326 advanced cases (106 cases were newly found in 2011), and no acute cases were found. About 24 454.78 hm2 of areas with Oncomelania snails were found in 2011, including 23 045.70 hm2 in marshland and lake regions (94.24%) and 1 409.08 hm2 in hilly region (5.76%). A total of 25 601 head of cattle were raised in schistosomiasis transmission regions in 2011, with a little increase comparing to that in 2010 (0.60%). The schistosome infection rate of cattle in 2011 (0.97%) decreased obviously comparing to that in 2010(3.52%). The chemotherapy coverage in cattle in 2011 (76.00%) increased significantly as compared to that in 2010 (50.85%) (P < 0.05). CONCLUSIONS: The schistosomiasis endemic situation remains at a certain level in endemic areas of Jiujiang City. The number of domestic animals is large, so the pollution of environment still exists. Therefore, it is suggested that promoting the comprehensive schistosomiasis control strategy with emphasis on controlling sources of infection is an effective measure to achieve the mid and long term goals of schistosomiasis control program.

Preparation of living isolated vertebrate photoreceptor cells for fluorescence imaging.

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells¹â»4. Rod photoreceptors are responsible for vision in dim light, cones in bright light. Phototransduction takes place in the outer segment of the photoreceptor cell, a specialized compartment that contains a high concentration of visual pigment, the primary light detector. The visual pigment is composed of a chromophore, 11-cis retinal, attached to a protein, opsin. A photon absorbed by the visual pigment isomerizes the chromophore from 11-cis to all-trans. This photoisomerization brings about a conformational change in the visual pigment that initiates a cascade of reactions culminating in a change in membrane potential, and bringing about the transduction of the light stimulus to an electrical signal. The recovery of the cell from light stimulation involves the deactivation of the intermediates activated by light, and the reestablishment of the membrane potential. Ca²+ modulates the activity of several of the enzymes involved in phototransduction, and its concentration is reduced upon light stimulation. In this way, Ca²+ plays an important role in the recovery of the cell from light stimulation and its adaptation to background light. Another essential part of the recovery process is the regeneration of the visual pigment that has been destroyed during light-detection by the photoisomerization of its 11-cis chromophore to all-trans5⁻7. This regeneration begins with the release of all-trans retinal by the photoactivated pigment, leaving behind the apo-protein opsin. The released all-trans retinal is rapidly reduced in a reaction utilizing NADPH to all- trans retinol, and opsin combines with fresh 11-cis retinal brought into the outer segment to reform the visual pigment. All-trans retinol is then transferred out of the outer segment and into neighboring cells by the specialized carrier Interphotoreceptor Retinoid Binding Protein (IRBP). Fluorescence imaging of single photoreceptor cells can be used to study their physiology and cell biology. Ca²+-sensitive fluorescent dyes can be used to examine in detail the interplay between outer segment Ca²+ changes and response to light8⁻¹² as well as the role of inner segment Ca²+ stores in Ca²+ homeostasis¹³â»¹4. Fluorescent dyes can also be used for measuring Mg² concentration¹5, pH, and as tracers of aqueous and membrane compartments¹6. Finally, the intrinsic fluorescence of all-trans retinol (vitamin A) can be used to monitor the kinetics of its formation and removal in single photoreceptor cells¹7⁻¹9.