Vitamin A aldehyde-taurine adducts function in photoreceptor cells.

To facilitate the movement of retinoids through the visual cycle and to limit nonspecific chemical reaction, multiple mechanisms are utilized to handle these molecules when not contained within the binding pocket of opsin. Vitamin A aldehyde is sequestered by reversible Schiff base formation with phosphatidylethanolamine (PE) and subsequently undergoes NADPH-dependent reduction. Otherwise inefficient handling of retinaldehyde can lead to the formation of fluorescent di-retinal compounds within the outer segments of photoreceptor cells. These bisretinoid fluorophores initiate photooxidative processes having adverse consequences for retina. Various carrier proteins confer water solubility and maintain the 11-cis-retinoid configuration. Mechanisms for sequestration of retinoid include the formation of a reversible Schiff base between retinaldehyde and taurine (A1-taurine, A1T), the most abundant amino acid in photoreceptor cells. Here we have undertaken to examine the effects of taurine depletion using the transport inhibitors guanidinoethyl sulfonate (GES) and ß-alanine. Oral treatment of BALB/cJ mice with ß-alanine reduced ocular A1T and the mice exhibited significantly lower scotopic and photopic a-wave amplitudes. As a secondary effect of retinal degeneration, A1T was not detected and taurine was significantly reduced in mice carrying a P23H opsin mutation. The thinning of ONL that is indicative of reduced photoreceptor cell viability in albino Abca4-/- mice was more pronounced in ß-alanine treated mice. Treatment of agouti and albino Abca4-/- mice with ß-alanine and GES was associated with reduced bisretinoid measured chromatographically. Consistent with a reduction in carbonyl scavenging activity by taurine, methylglyoxal-adducts were also increased in the presence of ß-alanine. Taken together these findings support the postulate that A1T serves as a reservoir of vitamin A aldehyde, with diminished A1T explaining reduced photoreceptor light-sensitivity, accentuated ONL thinning in Abca4-/- mice and attenuated bisretinoid formation.

Tissue and serum metabolomic phenotyping for diagnosis and prognosis of hepatocellular carcinoma.

More than two-thirds of patients with hepatocellular carcinoma (HCC) cannot receive curative therapy and have poor survival due to late diagnosis and few prognostic directions. In our study, nontargeted and targeted metabolomics analyses were conducted by liquid chromatography-mass spectrometry to characterize metabolic features of HCC and identify diagnostic and prognostic biomarker candidate incorporating liver tissue and serum metabolites. A total of 552 subjects, including 432 with liver tissue and 120 with serum specimens, were recruited in China. In the discovery cohort, a series of 138 metabolites were identified to discriminate HCC tissues from matched nontumor tissues. Retinol presented with the highest area under the curve (AUC) of 0.991 and associated with Edmondson grade. In the validation cohort, all metabolites in retinol metabolism pathway were examined and the levels of retinol and retinal in tumor tissue and serum decreased in the order of normal to cirrhosis to HCC of Edmondson Grades I to IV. Retinol and retinal levels could also differentiate between HCC and cirrhosis, with AUCs of 0.996 and 0.994, respectively, in tissue and 0.812 and 0.744, respectively, in serum. The AUC of the combined retinol and retinal panel in serum was 0.852. Univariate and multivariate Cox regression identified this panel as an independent predictor for HCC and showed that low expression of retinol and retinal correlated with decreased survival time. In conclusion, the retinol metabolic signature had considerable diagnostic and prognostic value for identifying HCC patients who would benefit from prompt therapy and optimal prognostic direction.

Retinyl esters are elevated in progeny of retinol dehydrogenase 11 deficient dams.

Retinol dehydrogenase 11 (RDH11) is an NADPH-dependent retinaldehyde reductase that was previously reported to function in the visual cycle. Recently, we have shown that RDH11 contributes to the maintenance of retinol levels in extraocular tissues under conditions of vitamin A deficiency or reduced vitamin A availability. RDH11 is also expressed in the embryo. Rdh11 knockout animals do not display embryonic defects and appear to develop normally to the adult stage, but the exact function of RDH11 during development is not yet known. In contrast to RDH11-null mice, animals that lack dehydrogenase/reductase 3 (DHRS3), the enzyme that functions as a retinaldehyde reductase and is essential for the maintenance of retinoid homeostasis during embryogenesis, rarely survive until birth. Here, we investigated whether inactivation of RDH11 together with DHRS3 exacerbates the severity of retinoid homeostasis disruption in embryos that lack both enzymes compared to DHRS3-null mice. The results of this study indicate that in vitamin A sufficient animals, the loss of RDH11 in addition to DHRS3 does not appear to significantly impact the total levels of retinoic acid, free retinol, or retinyl esters in Rdh11-/-/Dhrs3-/-embryos in comparison to Dhrs3-/- embryos. Surprisingly, Rdh11-/- single gene knockout embryos obtained from breeding of Rdh11-/- dams display elevated levels of embryonic retinyl esters compared to wild type embryos. The mechanism of the maternal effect of Rdh11 status on fetal retinoid stores remains to be elucidated.

Retinal Pre-Conditioning by CD59a Knockout Protects against Light-Induced Photoreceptor Degeneration.

Complement dysregulation plays a key role in the pathogenesis of age-related macular degeneration (AMD), but the specific mechanisms are incompletely understood. Complement also potentiates retinal degeneration in the murine light damage model. To test the retinal function of CD59a, a complement inhibitor, CD59a knockout (KO) mice were used for light damage (LD) experiments. Retinal degeneration and function were compared in WT versus KO mice following light damage. Gene expression changes, endoplasmic reticulum (ER) stress, and glial cell activation were also compared. At baseline, the ERG responses and rhodopsin levels were lower in CD59aKO compared to wild-type (WT) mice. Following LD, the ERG responses were better preserved in CD59aKO compared to WT mice. Correspondingly, the number of photoreceptors was higher in CD59aKO retinas than WT controls after LD. Under normal light conditions, CD59aKO mice had higher levels than WT for GFAP immunostaining in Müller cells, mRNA and protein levels of two ER-stress markers, and neurotrophic factors. The reduction in photon capture, together with the neurotrophic factor upregulation, may explain the structural and functional protection against LD in the CD59aKO.

Quantitation of retinaldehyde in small biological samples using ultrahigh-performance liquid chromatography tandem mass spectrometry.

We report an ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to quantify all-trans-retinal in biological samples of limited size (15-35mg), which is especially advantageous for use with adipose. To facilitate recovery, retinal and the internal standard 3,4-didehydroretinal were derivatized in situ into their O-ethyloximes. UHPLC resolution combined with high sensitivity and specificity of MS/MS allowed quantification of retinal-O-ethyloximes with a 5-fmol lower limit of detection and a linear range from 5fmol to 1pmol. This assay revealed that extraocular concentrations of retinal range from approximately 2 to 40pmol/g in multiple tissues-the same range as all-trans-retinoic acid. All-trans-retinoic acid has high affinity (kd⩽0.4nM) for its nuclear receptors (RARα, -ß, and -γ), whereas retinal has low (if any) affinity for these receptors, making it unlikely that these retinal concentrations would activate RAR. We also show that the copious amount of vitamin A used in chow diets increases retinal in adipose depots 2- to 5-fold relative to levels in adipose of mice fed a vitamin A-sufficient diet, as recommended for laboratory rodents. This assay also is proficient for quantifying conversion of retinol into retinal in vitro and, therefore, provides an efficient method to study metabolism of retinol in vivo and in vitro.

Effects of organic solvents on two retinal pigment epithelial lipofuscin fluorophores, A2E and all-trans-retinal dimer.

Gene and drug therapies are being developed to alleviate vision loss in patients with Stargardt's disease and age-related macular degeneration (AMD). To evaluate the therapeutic effects of these treatments, organic solvents are routinely used to extract and quantify bisretinoid lipofuscin constituents, such as N-retinylidene-N-retinyl-ethanolamine (A2E) and all-trans-retinal dimer (ATR-dimer). By high-performance liquid chromatography (HPLC), we found that A2E and ATR-dimer were both altered by tetrahydrofuran (THF) and chloroform, but were stable in dimethyl sulfoxide (DMSO) or methanol (MeOH). In addition, cyclohexane and ethanol (EtOH) did not alter ATR-dimer, whereas an alteration of A2E occurred in EtOH. On the basis of these findings, we designed processes II-IV, generated by modifications of process I, a routine method to measure bisretinoid compounds in vivo. Extra amounts of either ATR-dimer or A2E in mouse eyecups were released by processes II-IV versus process I. Efforts to clarify the effects of organic solvents on lipofuscin pigments are important because such studies can guide the handling of these fluorophores in related experiments.

Determination and occurrence of retinoids in a eutrophic lake (Taihu Lake, China): cyanobacteria blooms produce teratogenic retinal.

Besides retinoic acids (RAs), some retinoids such as retinal (RAL) and retinol (ROH), which are considered as RA precursors in vertebrates, are also reported to be teratogenic agents. In this study we investigated four RA precursors including RAL, ROH, retinyl palmitate, and ß-carotene in the eutrophic Taihu Lake, China, by developing a sensitive analytical method. RAL and ß-carotene were widely detected in natural cyanobacteria blooms and lake water. Intracellular concentrations of RAL and ß-carotene in blooms were 9.4 to 6.9 × 10(3) and 3.4 to 1.8 × 10(5) ng L(-1), respectively, and their concentrations in lake water were up to 1.4 × 10 ng L(-1) (RAL) and 9.8 × 10(2) ng L(-1) (ß-carotene). The good correlation between intracellular concentrations of RAL and RAs implied that RAL was involved in the production of RAs by cyanobacteria blooms. Further examination of 39 cyanobacteria and algae species revealed that most species could produce RAL and ß-carotene. The greatest amount of RAL was found in Chlamydomonas sp. (FACHB-715; 1.9 × 10(3) ng g(-1) dry weight). As the main cyanobacteria in Taihu Lake, many Microcystis species could produce high amounts of RAL and were thought to greatly contribute to the production of RAL measured in the blooms. Productions of RAL and ß-carotene by cyanobacteria were associated with species, origin location, and growth stage. The results in this study present the existence of a potential risk to aquatic animals living in a eutrophic environment from a high concentration of RAL in cyanobacteria blooms and also provide a clue for further investigating the mechanism underlying the biosynthetic pathway of RAs in cyanobacteria and algae.

Increased cone sensitivity to ABCA4 deficiency provides insight into macular vision loss in Stargardt's dystrophy.

Autosomal recessive Stargardt macular dystrophy is caused by mutations in the photoreceptor disc rim protein ABCA4/ABCR. Key clinical features of Stargardt disease include relatively mild rod defects such as delayed dark adaptation, coupled with severe cone defects reflected in macular atrophy and central vision loss. In spite of this clinical divergence, there has been no biochemical study of the effects of ABCA4 deficiency on cones vs. rods. Here we utilize the cone-dominant Abca4(-/-)/Nrl(-/-) double knockout mouse to study this issue. We show that as early as post-natal day (P) 30, Abca4(-/-)/Nrl(-/-) retinas have significantly fewer rosettes than Abca4(+/+)/Nrl(-/-) retinas, a phenotype often associated with accelerated degeneration. Abca4-deficient mice in both the wild-type and cone-dominant background accumulate more of the toxic bisretinoid A2E than their ABCA4-competent counterparts, but Abca4(-/-)/Nrl(-/-) eyes generate significantly more A2E per mole of 11-cis-retinal (11-cisRAL) than Abca4(-/-) eyes. At P120, Abca4(-/-)/Nrl(-/-) produced 340 ± 121 pmoles A2E/nmol 11-cisRAL while Abca4(-/-) produced 50.4 ± 8.05 pmoles A2E/nmol 11-cisRAL. Nevertheless, the retinal pigment epithelium (RPE) of Abca4(-/-)/Nrl(-/-) eyes exhibits fewer lipofuscin granules than the RPE of Abca4(-/-) eyes; at P120: Abca4(-/-)/Nrl(-/-) exhibit 0.045 ± 0.013 lipofuscingranules/µm² of RPE vs. Abca4(-/-) 0.17 ± 0.030 lipofuscingranules/µm² of RPE. These data indicate that ABCA4-deficient cones simultaneously generate more A2E than rods and are less able to effectively clear it, and suggest that primary cone toxicity may contribute to Stargardt's-associated macular vision loss in addition to cone death secondary to RPE atrophy.

HPLC/UV quantitation of retinal, retinol, and retinyl esters in serum and tissues.

We report robust HPLC/UV methods for quantifying retinyl esters (RE), retinol (ROL), and retinal (RAL) applicable to diverse biological samples with lower limits of detection of 0.7, 0.2, and 0.2 pmol, respectively, and linear ranges greater than 3 orders of magnitude. These assays function well with small, complex biological samples (10-20mg tissue). Coefficients of variation range from 5.9 to 10.0% (intraday) and from 5.9 to 11.0% (interday). Quantification of endogenous RE, ROL, and RAL in mouse serum and tissues (liver, kidney, adipose, muscle, spleen, testis, skin, brain, and brain regions) reveals utility. Ability to discriminate spatial concentrations of ROL and RE is illustrated with C57BL/6 mouse brain loci (hippocampus, cortex, olfactory bulb, thalamus, cerebellum, and striatum). We also developed a method to distinguish isomeric forms of ROL to investigate precursors of retinoic acid. The ROL isomer assay has limits of detection between 3.5 and 4.5 pmol and has a linear range and coefficient of variation similar to those of the ROL/RE and RAL assays. The assays described here provide for sensitive and rigorous quantification of endogenous RE, ROL, and RAL to elucidate retinoid homeostasis in disease states such as Alzheimer's disease, type 2 diabetes, obesity, and cancer.

Alpha-retinals as rhodopsin chromophores--preference for the 9-Z configuration and partial agonist activity.

The visual pigment rhodopsin, the photosensory element of the rod photoreceptor cell in the vertebrate retina, shows in combination with an endogenous ligand, 11-Z retinal, an astonishing photochemical performance. It exhibits an unprecedented quantum yield (0.67) in a highly defined and ultrafast photoisomerization process. This triggers the conformational changes leading to the active state Meta(rhodopsin) II. Retinal is covalently bound to Lys-296 of the protein opsin in a protonated Schiff base. The resulting positive charge delocalization over the terminal part of the polyene chain of retinal creates a conjugation defect that upon photoexcitation moves to the opposite end of the polyene. Shortening the polyene as in 4,5-dehydro,5,6-dihydro (alpha), 5,6-dihydro or 7,8-dihydro-analogs might facilitate photoisomerization of a 9-Z and a 11-Z bond. Here we describe pigment analogs generated with bovine opsin and 11-Z or 9-Z 4,5-dehydro,5,6-dihydro-retinal that were further characterized by UV-Vis and FTIR spectroscopy. The preference of opsin for native 11-Z retinal over the 9-Z isomer is reversed in 4,5-dehydro,5,6-dihydro-retinal. 9-Z 4,5-dehydro,5,6-dihydro-retinal readily generated a photosensitive pigment. This modification has no effect on the quantum yield, but affects the Batho<-->blueshifted intermediate (BSI) equilibrium and leads to a strong decrease in the G-protein activation rate because of a downshift of the pK(a) of the Meta I<-->Meta II equilibrium.

The all-trans-retinal dimer series of lipofuscin pigments in retinal pigment epithelial cells in a recessive Stargardt disease model.

The bis-retinoid pigments that accumulate in retinal pigment epithelial cells as lipofuscin are associated with inherited and age-related retinal disease. In addition to A2E and related cis isomers, we previously showed that condensation of two molecules of all-trans-retinal leads to the formation of a protonated Schiff base conjugate, all-trans-retinal dimer-phosphatidylethanolamine. Here we report the characterization of the related pigments, all-trans-retinal dimer-ethanolamine and unconjugated all-trans-retinal dimer, in human and mouse retinal pigment epithelium. In eyecups of Abcr(-/-) mice, a model of recessive Stargardt macular degeneration, all-trans-retinal dimer-phosphatidylethanolamine was increased relative to wild type and was more abundant than A2E. Total pigment of the all-trans-retinal dimer series (sum of all-trans-retinal dimer-phosphatidylethanolamine, all-trans-retinal dimer-ethanolamine, and all-trans-retinal dimer) increased with age in Abcr(-/-) mice and was modulated by amino acid variants in Rpe65. In in vitro assays, enzyme-mediated hydrolysis of all-trans-retinal dimer-phosphatidylethanolamine generated all-trans-retinal dimer-ethanolamine, and protonation/deprotonation of the Schiff base nitrogen of all-trans-retinal dimer-ethanolamine was pH-dependent. Unconjugated all-trans-retinal dimer was a more efficient generator of singlet oxygen than A2E, and the all-trans-retinal dimer series was more reactive with singlet oxygen than was A2E. By analyzing chromatographic properties and UV-visible spectra together with mass spectrometry, mono- and bis-oxygenated all-trans-retinal dimer photoproducts were detected in Abcr(-/-) mice. The latter findings are significant to an understanding of the adverse effects of retinal pigment epithelial cell lipofuscin.

Cantilever-based sensor for the detection of different chromophore isomers.

We report the use of microcantilevers (MCs) for the detection of three retinoid isomers: 9-cis-retinal, 13-cis-retinal and all-trans-retinal. Detection of synthetic and natural retinoids in topical cosmetic products is important, and their presence can be used to predict reactions with the skin surface. In this study the MC surfaces were functionalized in order to promote the formation of covalent bonds with the chromophores. The lowest mass shift we detected with the functionalized MCs was 1.2 ppt, which is in the range needed by the cosmetics industry. Our results indicate that properly designed and functionalized microcantilevers can be used to construct economical, fast, and sensitive sensors for quality control in cosmetics.

The effects of copper and benzo[a]pyrene on retinoids and reproduction in zebrafish.

This study examines whether a link exists between toxicant exposure, retinoids and reproduction in fish. Zebrafish were fed a control diet (8.1 microg Cu/g diet, 0 microg benzo[a]pyrene/g diet) or diets containing elevated copper (100 microg, 500 microg and 1000 microg Cu/g diet) or benzo[a]pyrene (B[a]P; 30 and 150 microg B[a]P/g diet) for 260 days. Toxicant-supplemented diets did not affect growth or mortality rates. While whole body retinoid levels in control zebrafish decreased during the experiment, females exposed to Cu or B[a]P for 200 days or more experienced additional losses of retinyl esters (45-100% depleted) and retinal (45% depleted in B[a]P-fed fish). Despite the reduced retinoids, Cu and B[a]P did not effect reproduction with respect to the number of eggs spawned, fertilization rates or egg retinal content (retinal was instead increased 55-65% in eggs from B[a]P-fed fish). There were no apparent deformities observed in 36 h post fertilization embryos from any treatment. It appears that although internal retinoid stores were depleted in adults, dietary retinoids were sufficient to meet the daily requirement for retinal deposition in the eggs and retinoic acid synthesis. This study has shown that retinoid levels in female zebrafish are sensitive to Cu and B[a]P, and are a good indicator of long-term exposure. It also brings to light the resiliency of the retinoid system in fish and the importance of the diet on the toxicological response. Specifically that dietary retinoids appear to support normal reproduction in the absence of internal retinoid stores.

Retinal biosynthesis in fungi: characterization of the carotenoid oxygenase CarX from Fusarium fujikuroi.

The car gene cluster of the ascomycete Fusarium fujikuroi encodes two enzymes responsible for torulene biosynthesis (CarRA and CarB), an opsin-like protein (CarO), and a putative carotenoid cleaving enzyme (CarX). It was presumed that CarX catalyzes the formation of the major carotenoid in F. fujikuroi, neurosporaxanthin, a cleavage product of torulene. However, targeted deletion of carX did not impede neurosporaxanthin biosynthesis. On the contrary, DeltacarX mutants showed a significant increase in the total carotenoid content, indicating an involvement of CarX in the regulation of the pathway. In this work, we investigated the enzymatic activity of CarX. The expression of the enzyme in beta-carotene-accumulating Escherichia coli cells led to the formation of the opsin chromophore retinal. The identity of the product was proven by high-performance liquid chromatography and gas chromatography-mass spectrometry. Subsequent in vitro assays with heterologously expressed and purified CarX confirmed its beta-carotene-cleaving activity and revealed its capability to produce retinal also from other substrates, such as gamma-carotene, torulene, and beta-apo-8'-carotenal. Our data indicate that the occurrence of at least one beta-ionone ring in the substrate is required for the cleavage reaction and that the cleavage site is determined by the distance to the beta-ionone ring. CarX represents the first retinal-synthesizing enzyme reported in the fungal kingdom so far. It seems likely that the formed retinal is involved in the regulation of the carotenoid biosynthetic pathway via a negative feedback mechanism.

Imaging the dynamic behaviour of individual retinal chromophores confined inside carbon nanotubes.

Retinal is the molecule found in photoreceptor cells that undergoes a change in shape when it absorbs light. Specifically, the cis/trans isomerization of a carbon-carbon double bond in this chromophore sets in motion the chain of biochemical processes responsible for vision. Here, we obtain atomically resolved images of individual structural isomers of the retinal chromophore attached to C60 molecules and study their dynamic behaviour inside a confined space--that is, inside single-walled carbon nanotubes--using high-resolution transmission electron microscopy (HR-TEM). Sequential HR-TEM images with sub-second time resolution directly reveal the isomerization between the cis and all-trans forms of retinal, as well as conformational changes and volume-conserving effects. This work opens up the possibility of investigating in vitro the biological activities of these photoresponsive molecules on an individual basis, and the molecular imaging technique described here is a general one that can be applied to a wide range of systems.

Light-induced damage to the retina: role of rhodopsin chromophore revisited.

The presence of the regenerable visual pigment rhodopsin has been shown to be primarily responsible for the acute photodamage to the retina. The photoexcitation of rhodopsin leads to isomerization of its chromophore 11-cis-retinal to all-trans-retinal (ATR). ATR is a potent photosensitizer and its role in mediating photodamage has been suspected for over two decades. However, there was lack of experimental evidence that free ATR exists in the retina in sufficient concentrations to impose a risk of photosensitized damage. Identification in the retina of a retinal dimer and a pyridinium bisretinoid, so called A2E, and determination of its biosynthetic pathway indicate that substantial amounts of ATR do accumulate in the retina. Both light damage and A2E accumulation are facilitated under conditions where efficient retinoid cycle operates. Efficient retinoid cycle leads to rapid regeneration of rhodopsin, which may result in ATR release from the opsin "exit site" before its enzymatic reduction to all-trans-retinol. Here we discuss photodamage to the retina where ATR could play a role as the main toxic and/or phototoxic agent. Moreover, we discuss secondary products of (photo)toxic properties accumulating within retinal lipofuscin as a result of ATR accumulation.

Toward the active conformations of rhodopsin and the beta2-adrenergic receptor.

Using sets of experimental distance restraints, which characterize active or inactive receptor conformations, and the X-ray crystal structure of the inactive form of bovine rhodopsin as a starting point, we have constructed models of both the active and inactive forms of rhodopsin and the beta2-adrenergic G-protein coupled receptors (GPCRs). The distance restraints were obtained from published data for site-directed crosslinking, engineered zinc binding, site-directed spin-labeling, IR spectroscopy, and cysteine accessibility studies conducted on class A GPCRs. Molecular dynamics simulations in the presence of either "active" or "inactive" restraints were used to generate two distinguishable receptor models. The process for generating the inactive and active models was validated by the hit rates, yields, and enrichment factors determined for the selection of antagonists in the inactive model and for the selection of agonists in the active model from a set of nonadrenergic GPCR drug-like ligands in a virtual screen using ligand docking software. The simulation results provide new insights into the relationships observed between selected biochemical data, the crystal structure of rhodopsin, and the structural rearrangements that occur during activation.

Effect of beta-particles on the retinal chromophore in bacteriorhodopsin of Halobacterium salinarium.

Bacteriorhodopsin (bR) is an attractive intelligent material. Understanding the mechanism of its light-driven proton pumping outward the cell implicates it in many technical applications, particularly, in what is called optical computers, and the biotechnology is waiting for this promised biological molecule. An ionizing radiation source handling could be computerized in radiation fields. The computer containing such biological material will not be out of reach of the fields of ionizing radiation. So it is interesting to report on the working of such biological computer if it is subjected to ionizing radiation. The functional unit in this molecule is retinal chromophore. In the present work, it is interested to assess the functionality of bR through determining the electronic transition dipole moment of its chromophore. Significant changes in the values of the absorption transition dipole moment were noticed at different doses of beta-particles in the range of 0.1-0.3 kGy. Ionizing radiation-induced changes in bR were followed by intrinsic fluorescence spectroscopy. An analysis of the fluorescence data bears on the tertiary structure of bR. The emission spectrum is, however, red shifted with an increase in intensity with the different doses; in the meanwhile, gradual decrease in the visible absorbance has occurred till almost complete loss is attained. This bleaching due to ionizing radiation may offer an alternative way of data processing in such optical devices based on bR. Nevertheless, bR has proofed to be used as a biological indicator of ionizing radiation. However, the potential of bR for use as a biosensor to detect ionizing radiation should be considered.

The photoreceptive capacity of the developing pineal gland and eye of the golden hamster (Mesocricetus auratus).

Anatomical and physiological studies have suggested that the pineal gland of neonatal mammals has a photoreceptive capacity. Using the golden hamster (Mesocricetus auratus) as our model, we applied biochemical approaches to look for a functional photopigment within the pineal during early development. Immunocytochemistry and enzyme-linked immunosorbent assay (ELISA) were used to localize and quantify opsin, and high-performance liquid chromatography (HPLC) to identify photopigment chromophore (11-cis and all-trans retinaldehyde) in the developing eye and pineal. For HPLC analysis, retinaldehydes were converted to their corresponding retinoid oximes. Eluted retinoids were identified by comparison with standard vitamin A1 retinoid oxime isomers on the basis of relative elution sequence and characteristic absorbance spectra. Both immunocytochemistry and ELISA suggested an increase in the opsin content of the pineal during the first week of life. In the eye, 11-cis retinaldehyde was first detected between days 3 and 5 after birth. In three separate extractions, and using a considerable excess of pineal tissue, we failed to identify chromophore within the pineal during the first week of postnatal development. The appearance of 11-cis retinaldehyde within the eye between postnatal days 3-5 is consistent with the hypothesis that retinol isomerase activity is coordinated with outer segment development. The failure to identify chromophore within the neonatal pineal suggests that this gland lacks a functional opsin-based photopigment. These data contradict physiological evidence suggesting that the neonatal pineal of mammals contains photoreceptors.