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Bistable UV pigment in the lamprey pineal.

Lower vertebrates can detect UV light with the pineal complex independently of eyes. Electrophysiological studies, together with chromophore extraction analysis, have suggested that the underlying pigment in the lamprey pineal exhibits a bistable nature, that is, reversible photoreaction by UV and visible light, which is never achieved by known UV pigments. Here we addressed the molecular identification of the pineal UV receptor. Our results showed that the long-hypothesized pigment is a lamprey homologue of parapinopsin, which exhibits an absorption maximum at 370 nm, in the UV region. UV light causes cis-trans isomerization of its retinal(2) chromophore, forming a stable photoproduct having an absorption maximum at 515 nm, in the green region. The photoproduct reverts to the original pigment upon visible light absorption, showing photoregeneration of the pigment. In situ hybridization showed that parapinopsin is selectively expressed in the cells located in the dorsal region of the pineal organ. We successfully obtained the hyperpolarizing responses with a maximum sensitivity of approximately 380 nm from the photoreceptor cells at the dorsal region, in which the outer segment was clearly stained with anti-parapinopsin antibody. These results demonstrated that parapinopsin is the pineal UV pigment having photointerconvertible two stable states. The bistable nature of the parapinopsin can account for the photorecovery of the pineal UV sensitivity by background green light in the lamprey. Furthermore, we isolated the parapinopsin homologues from fish and frog pineal complexes that exhibit UV sensitivity, suggesting that parapinopsin is a common molecular basis for pineal UV reception in the vertebrate.

Animals↗

Highly effective phosphorylation by G protein-coupled receptor kinase 7 of light-activated visual pigment in cones.

Cone photoreceptors show briefer photoresponses than rod photoreceptors. Our previous study showed that visual pigment phosphorylation, a quenching mechanism of light-activated visual pigment, is much more rapid in cones than in rods. Here, we measured the early time course of this rapid phosphorylation with good time resolution and directly compared it with the photoresponse time course in cones. At the time of photoresponse recovery, almost two phosphates were incorporated into a bleached cone pigment molecule, which indicated that the visual pigment phosphorylation coincides with the photoresponse recovery. The rapid phosphorylation in cones is attributed to very high activity of visual pigment kinase [G protein-coupled receptor kinase (GRK) 7] in cones. Because of this high activity, cone pigment is readily phosphorylated at very high bleach levels, which probably explains why cone photoresponses recover quickly even after a very bright light and do not saturate under intense background light. The high GRK7 activity is brought about by high content of a highly potent enzyme. The expression level of GRK7 was 10 times higher than that of rod kinase (GRK1), and the specific activity of a single GRK7 molecule was approximately 10 times higher than that of GRK1. The specific activity of GRK7 is the highest among the GRKs so far known. Our result seems to explain the response characteristics of cone photoreceptors in many aspects, including the nonsaturation of the cone responses during daylight vision.

Animals↗

Isorhodopsin II: artificial photosensitive pigment formed from 9,13-dicis retinal.

We have found that in addition to the 11-cis and 9-cis isomers of retinal which are known to couple with the visual pigment apoprotein opsin to form pigments, a third isomer 9,13-dicis retinal also will form a pigment. That this isomer is indeed bound to opsin has been shown unequivocally by removing the chromophore without isomerization and subsequent identification by high-speed liquid chromatography. Using similar techniques, we have shown that the product of bleaching by light of all three pigments in Trition X-100 is the all-trans isomer. This specificity in the product of bleaching, as with many other properties of visual pigments, is not shared by the free chromophore. Of particular interest is that when 9,13-dicis retinal is combined with opsin to form a pigment, a single photon can isomerize it about two double bonds, to the all-trans isomer.

Binding Sites↗

Anion sensitivity and spectral tuning of cone visual pigments in situ.

We tested the effect of anions on the absorbance spectrum of native visual pigments as measured by microspectrophotometry in individual cone outer segments of four species of fish and one species of amphibian. In all species tested, the long-wavelength-absorbing cone pigments were anion sensitive, and their lambda max could be tuned over a range of 55 nm depending on the identity of the anion present. Cl- and Br- were the only anions that produced native pigment spectra by red shifting lambda max from its value under anion-free conditions. Lyotropic anions such as NO3-, SCN-, BF4-, and ClO4- caused substantial and graded blue shifts of lambda max. The apparent Kd of binding sites on the pigment for Cl- and for ClO4- was approximately 2 mM. Taken together with previous findings on three visual pigments from the reptilian, avian, and amphibian classes, our results support the hypothesis that all long-wavelength-absorbing vertebrate visual pigments are spectrally tuned in part through the binding of a chloride ion. We propose that the site of anion tuning is near the protonated Schiff base of the chromophore, whose counterion may be complex and include Cl- as an exchangeable anion. This counterion configuration may resemble the one present in the light-driven Cl- pump halorhodopsin.

Ambystoma↗

A fully functional rod visual pigment in a blind mammal. A case for adaptive functional reorganization?

In the blind subterranean mole rat Spalax ehrenbergi superspecies complete ablation of the visual image-forming capability has been accompanied by an expansion of the bilateral projection from the retina to the suprachiasmatic nucleus. We have cloned the open reading frame of a visual pigment from Spalax that shows >90% homology with mammalian rod pigments. Baculovirus expression yields a membrane protein with all functional characteristics of a rod visual pigment (lambda(max) = 497 +/- 2 nm; pK(a) of meta I/meta II equilibrium = 6.5; rapid activation of transducin in the light). We not only provide evidence that this Spalax rod pigment is fully functional in vitro but also show that all requirements for a functional pigment are present in vivo. The physiological consequences of this unexpected finding are discussed. One attractive option is that during adaptation to a subterranean lifestyle, the visual system of this mammal has undergone mosaic reorganization, and the visual pigments have adapted to a function in circadian photoreception.

Amino Acid Sequence↗

Skin pigmentation and texture changes after hair removal with the normal-mode ruby laser.

Promising clinical results have been obtained with the normal mode ruby laser for removal of unwanted hair. Melanin within the hair follicles is thought to act as target for the ruby laser pulses, whereas epidermal melanin is thought to be a competitive chromophore, responsible for potential side effects. This study aimed (i) to objectify postoperative changes in skin pigmentation and texture and (ii) to evaluate the importance of variations in preoperative skin pigmentation for the development of side effects 12 weeks after 1 treatment with the normal-mode ruby laser. A total of 17 volunteers (skin types I-IV) were laser-treated in the hairy pubic region (n = 51 test areas). A shaved test area served as control. Skin reflectance spectroscopical measurements, 3-dimensional surface contour analysis and ultrasonography objectified postoperative changes in skin pigmentation and texture. Blinded clinical assessments revealed postoperative hyperpigmentation (2% of test areas) and hypopigmentation (10%), whereas no textural changes were seen. Reflectance spectroscopically-determined pigmentary changes depended on the degree of preoperative skin pigmentation, fairly pigmented skin types experiencing subclinical hyperpigmentation and darkly pigmented skin types experiencing subclinical hypopigmentation. Three-dimensional surface profilometry documented similar pre- and postoperative surface contour parameters, indicating that the skin surface texture is preserved after laser exposure. Ultrasonography revealed similar skin thicknesses in laser-exposed and untreated control areas. It is concluded that normal-mode ruby laser treatment is safe for hair removal in skin types I-IV.

Adult↗

Effect of intensity of eggshell pigment and illuminated incubation on hatchability of brown eggs.

The effects of intensity of brown eggshell pigment (light (LBP), medium (MBP) and dark (DBP)) and light intensity during incubation (low and high, 900 to 1380 and 1430 to 2080 lux, respectively) on eggshell characteristics, embryonic growth, hatchability traits, chick hatching weight and hatching time were investigated using eggs from a meat-type breeder (Hybro) flock at 32, 36 and 41 weeks of age in three trials. With eggs of similar weights the intensity of brown pigment was not associated with eggshell weight and thickness, and did not influence embryo weight and egg weight loss during incubation. The shade of brown pigment of eggs laid by young hens influenced the percentage hatchability (HP) of eggs incubated under light. Illuminated incubation improved HP of LBP eggs (compared with MBP and DBP eggs) from 32- and 36-week-old hens, but had no significant effect on HP of eggs from 41-week-old hens. Light intensity during incubation did not influence egg weight loss. High intensity of light during incubation reduced HP and increased early death percentage (EDP) in the LBP and MBP groups, and did not influence HP and EDP in the DBP group. Brown eggshell pigment and intensity of light during incubation did not influence hatching time. It is concluded that the shade of brown pigment, intensity of light during incubation and age of the breeder hens influenced the hatchability performance of embryos from brown eggs. Light during incubation improved the hatchability of embryos in light brown eggs laid by young hens and the shade of brown pigment of eggs laid by older hens did not influence hatchability under illuminated incubation. High intensity of light during incubation reduced hatchability of light and medium brown eggs, but not the dark brown eggs.

Animals↗

Effects of phosphate, prostaglandins, arachidonic acid and arginine vasotocin on oviposition and pigment secretion from the shell gland in Japanese quail.

1. Phosphate solution, prostaglandin F2alpha and E2, arachidonic acid and arginine vasotocin were injected intrauterinely or intravenously into laying quail hens 6 h before expected oviposition. Following injections, induced ovipositions and secretions of pigment from their shell glands were observed. 2. The effects of intrauterine injection with prostaglandins, which induced oviposition and pigment secretion, were not inhibited by pre-injection of indomethacin. 3. Indomethacin completely inhibited the inducing effects on oviposition and pigment secretion of intrauterine injections with phosphate solution and arachidonic acid. 4. Intravenous injection with arginine vasotocin or prostaglandins also induced oviposition and pigment secretion; indomethacin, however, only inhibited pigment secretion. 5. It is concluded that the effects of prostaglandins on pigment secretion from the shell gland were demonstrated experimentally.

Animals↗

Determination of astaxanthin stereoisomers and colour attributes in flesh of rainbow trout (Oncorhynchus mykiss) as a tool to distinguish the dietary pigmentation source.

The presence of carotenoids in animal tissue reflects their sources along the food chain. Astaxanthin, the main carotenoid used for salmonid pigmentation, is usually included in the feed as a synthetic product. However, other dietary sources of astaxanthin such as shrimp or krill wastes, algae meal or yeasts are also available on the market. Astaxanthin possesses two identical asymmetric atoms at C-3 and C-3' making possible three optical isomers with all-trans configuration of the chain: 3S,3'S, 3R,3'S, and 3R,3'R. The distribution of the isomers in natural astaxanthin differs from that of the synthetic product. This latter is a racemic mixture, with a typical ratio of 1:2:1 (3S,3'S:3R,3'S:3R,3'R), while astaxanthin from natural sources has a variable distribution of the isomers deriving from the different biological organism that synthesized it. The high-performance liquid chromatographic (HPLC) analysis of all-trans isomers of astaxanthin was performed in different pigment sources, such as red yeast Phaffia rhodozyma, alga meal Haematococcus pluvialis, krill meal and oil, and shrimp meal. With the aim to investigate astaxanthin isomer ratios in flesh of fish fed different carotenoid sources, three groups of rainbow trout were fed for 60 days diets containing astaxanthin from synthetic source, H. pluvialis algae meal and P. rhodozyma red yeast. Moreover, the distribution of optical isomers of astaxanthin in trout purchased on the Italian market was investigated. A characteristic distribution of astaxanthin stereoisomers was detected for each pigment sources and such distribution was reproduced in the flesh of trout fed with that source. Colour values measured in different sites of fillet of rainbow trout fed with different pigment sources showed no significant differences. Similarly, different sources of pigment (natural or synthetic) produced colour values of fresh fillet with no relevant or significant differences. The coefficient of distance computed amongst the feed ingredient and the trout fillet astaxanthin stereoisomers was a useful tool to identify the origin of the pigment used on farm.

Animals↗

Uveal melanocytes, ocular pigment epithelium, and Müller cells in culture: in vitro toxicology.

Uveal melanocytes and the ocular pigment epithelium are located in the middle and inner layers of the eye. Müller cells (a type of glial cell) are located in the neural retina. Melanocytes, retinal pigment epithelium (RPE), and Müller cells do not participate directly in the detection or transfer of visual information, but they have various functions that support the neural retina and are essential for the maintenance of vision. Methods for the isolation and cultivation of melanocytes, RPE, and Müller cells have been established by us and other investigators. These cultured cells can be used as in vitro model systems for studying the toxicology of visible light, ultraviolet (UV) radiation, drugs, and other potentially toxic agents. Toxic effects on these cells may give rise to altered retinal function and result in impaired vision. Both melanocytes and pigment epithelium contain melanin, which has the ability to bind organic amines and metal ions. This results in the accumulation of these substances in the eye. Melanin may protect cells from chemical stress by binding toxic chemicals; but in chronic exposure, increased and lengthy binding may cause damage to these cells. Two different types of melanin are found in the eye: eumelanin and pheomelanin, which may have photoprotective and phototoxic effects, respectively. Pigment epithelium contains mainly eumelanin, whereas melanocytes contain both eumelanin and pheomelanin. Melanin is an antioxidant and with age, the antioxidant properties may diminish to the point that it may even become a prooxidant. There are also other functions of pigment epithelium and uveal melanocytes not related to melanin and there are also several functions of Muller cells that play a role in the toxicological aspects of the eye. Cultured uveal melanocytes, pigment epithelial cells, and Müller cells can be used to study the toxicology of these cells in vitro.

Animal Testing Alternatives↗

Q-switched ruby laser irradiation on spotty pigmentation in the skin of the hairless dog.

OBJECTIVE: To investigate macroscopically and histopathologically the dermatological changes after Q-switched ruby laser (QRL) irradiation with different exposure doses in UVB-induced pigmentation in hairless dogs. METHODS: QRL irradiation with 3.0, 5.0 and 7.0 J/cm(2) was carried out on the UVB-induced spotty pigmentation in the skin of the hairless dog. Gross appearance was observed daily throughout this study. Histopathological examination was performed 1 day before QRL irradiation and 1 and 3 days and 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14 and 16 weeks after QRL irradiation. RESULTS: Immediately after QRL irradiation, spotty pigmentation was removed. One week after QRL irradiation, re-epithelialization started from the margin of the irradiated sites. between 5 and 10 weeks after QRL irradiation, the skin color returned to normal and some portions showed recurrence of hyperpigmentation. Histopathologically, spotty pigmentation had a heavy deposition of melanin granules in the stratum basale, spinosum and corneum. One day after QRL irradiation, the skin showed destruction of melanin granules. Seven days after QRL irradiation, re-epithelialization began from the surroundings of the QRL-irradiated sites and the pilosebaceous units. The delayed process of re-epithelialization was dependent on the incident exposure dose with QRL. The repaired epidermis was devoid of melanin granules. By 5 weeks after QRL irradiation with 3.0 and 5.0 J/cm(2), the stratum basale and spinosum revealed a redistribution of melanin granules. In the sites of recurrent hyperpigmentation, the bases of the remaining hair follicles showed a notable increase in the reproduction of melanin granules. Melanin granules abundantly aggregated in the bottom portion of the nucleus in each epidermal cell. CONCLUSION: These results revealed that hairless dogs were invaluable laboratory animals, which developed spotty pigmentation after successive UVB irradiation. In addition, UVB-induced spotty pigmentation in hairless dogs is useful for investigating the process of depigmentory treatment with QRL irradiation and recurrence of this lesion.

Animals↗

Influence of pigment concentration and particle size on adhesion of an acrylic resin copolymer to tablet compacts.

The effects of the particle size and the concentration of pigments in aqueous polymeric dispersions on the adhesive properties of an acrylic resin copolymer were investigated. Aqueous polymeric dispersions containing up to 20% (v/v) pigment were coated onto hydrophilic and hydrophobic tablet compacts, and polymer adhesion was assessed using a novel butt adhesion technique. An inverse relationship was found between the particle size of the pigment present in the aqueous polymeric dispersion and film-tablet adhesion. As the particle size of the pigment increased, the adhesive strength of the polymer to the tablet compact decreased. Increased concentrations of the opacifying agent titanium dioxide in the acrylic dispersion resulted in stronger film-tablet adhesion. No clear relationship could be established between the wettability of the tablet compact by the pigmented polymeric dispersion and the strength of film-tablet adhesion. The hydrophobicity of the tablet compact was found to affect the glass transition temperature of the polymeric film to a greater extent than the particle size, morphology, or concentration of the pigment incorporated into the acrylic dispersion.

Acrylic Resins↗

Photoreceptor-pigment epithelial cell relationships in rats with inherited retinal degeneration. Radioautographic and electron microscope evidence for a dual source of extra lamellar material.

Protein synthesis and displacement in photoreceptor and pigment epithelial cells of inbred normal (Fisher) and mutant (RCS) rats with inherited retinal degeneration has been studied by light and electron microscope radioautography. Groups of animals 14, 15, 17, 19, 27, 35, and 50 days of age were injected with amino acids-H(3) and killed at subsequent time intervals. In normal rats, radioactive protein synthesized in the rod inner segments was incorporated into outer segment saccules and displaced outward; the total renewal time of outer segments at all ages was approximately 9 days. In RCS photoreceptors, outer segment displacement was slowed from the normal rate before day 17 and at all subsequent stages. Most of the newly synthesized protein appeared to migrate only into the basal third of the outer segments. Labeling of pigment epithelial cells in RCS rats was always heavier than in controls. Labeled protein was displaced as early as 1 hr postinjection from pigment epithelial cell somas into the apical processes, and by 2 hr postinjection was located in the adjacent lamellar whorls characteristic of the mutant rat retina. After 1 day, radioactivity was present in the 14, 15, 17, and 19 day series of RCS rats in the apical third of the outer segment layer (occupied mainly by extra lamellar material) while there were few silver grains in the middle third of the layer (occupied mainly by distal parts of outer segments). The RCS pigment epithelial cells thus have an unusual synthetic role and appear to be a source of the extra lamellar material. Electron microscope examination revealed that many intact pigment epithelial cell processes were incorporated into the large whorls of extra lamellae. In addition, many disorganized outer segment saccules were observed in continuity with longer membranous lamellae and large lamellar whorls. The extra lamellar material therefore appears to be derived from both rod outer segments and pigment epithelial cells.

Age Factors↗

Microspectrophotometry of arthropod visual screening pigments.

Absorption spectra of visual screening pigments obtained in vitro with a microspectrophotometer using frozen sections are given for the insects Musca domestica, Phormia regina, Libellula luctuosa, Apis mellifera (worker honeybee only), Drosophila melanogaster (wild type only) and the arachnids Lycosa baltimoriana and Lycosa miami. The spectral range covered is 260-700 nm for Lycosa and Drosophila and 310-700 nm for the remainder of the arthropods. A complete description of the instrumentation is given. For the flies, Phormia and Musca, light absorption by the yellow and red pigments is high from 310 to about 610 nm. This implies that for these insects there should be no wavelength shift in electroretinogram (ERG) results due to light leakage among neighboring ommatidia for this wavelength range. The same comment applies to Calliphora erythrocephala, which is known to have similar screening pigments. For some of the insects studied a close correspondence is noted between screening pigment absorption spectra and spectral sensitivity curves for individual photoreceptors, available in the literature. In some cases the screening pigment absorption spectra can be related to chemical extraction results, with the general observation that some of the in vitro absorption peaks are shifted to the red. The Lycosa, Apis, and Libellula dark red pigments absorb strongly over a wide spectral range and therefore prevent chemical identification.

Animals↗

Fast electrical potential from a long-lived, long-wavelength photoproduct of fly visual pigment.

A rapid electrical potential, which we have named the M-potential, can be obtained from the Drosophila eye using a high energy flash stimulus. The potential can be elicited from the normal fly, but it is especially prominent in the mutant norp A(P12) (a phototransduction mutant), particularly if the eye color pigments are genetically removed from the eye. Several lines of evidence suggest that the M-potential arises from photoexcitation of long-lived metarhodopsin. Photoexcitation of rhodopsin does not produce a comparable potential. The spectral sensitivity of the M-potential peaks at about 575 nm. The M-potential pigment (metarhodopsin) can be shown to photoconvert back and forth with a "silent pigment(s)" absorbing maximally at about 485 nm. The silent pigment presumably is rhodopsin. These results support the recent spectrophotometric findings that dipteran metarhodopsin absorbs at much longer wavelengths than rhodopsin. The M-potential probably is related to the photoproduct component of the early receptor potential (ERP). Two major differences between the M-potential and the classical ERP are: (a) Drosophila rhodopsin does not produce a rapid photoresponse, and (b) an anesthetized or freshly sacrificed animal does not yield the M-potential. As in the case of the ERP, the M-potential appears to be a response associated with a particular state of the fly visual pigment. Therefore, it should be useful in in vivo investigations of the fly visual pigment, about which little is known.

Animals↗

Adaptation of a deep-sea cephalopod to the photic environment. Evidence for three visual pigments.

Watasenia scintillans, a bioluminescent deep-sea squid, has a specially developed eye with a large open pupil and three visual pigments. Photoreceptor cells (outer segment: 476 micron; inner segment: 99 micron) were long in the small area of the ventral retina receiving downwelling light, whereas they were short (outer segment: 207 micron; inner segment: 44 micron) in the other regions of the retina. The short photoreceptor cells contained the visual pigment with retinal (lambda max approximately 484 nm), probably for the purpose of adapting to their environmental light. The outer segment of the long photoreceptor cells consisted of two strata, a pinkish proximal area and a yellow distal area. The visual pigment with 3-dehydroretinal (lambda max approximately 500 nm) was located in the pinkish proximal area, giving high sensitivity at longer wavelengths. A newly found pigment (lambda max approximately 471 nm) was in the yellow distal area. The small area of the ventral retina containing two visual pigments is thought to have a high and broad spectral sensitivity, which is useful for distinguishing the bioluminescence of squids of the same species in their environmental downwelling light. These findings were obtained by partial bleaching of the extracted pigment from various areas of the retina and by high-performance liquid chromatographic analysis of the chromophore, complemented by microscopic observations.

Adaptation, Physiological↗

Retinal pigment epithelial cell plasma membrane: a monoclonal antibody study.

The plasma membranes of retinal pigment epithelial cells are highly specialized organelles with multiple functions including nutritional and metabolic support of the photoreceptor cells. Using purified bovine retinal epithelial cell plasma membranes as antigen, we produced two monoclonal antibodies, MAbD1-C6 and MAbD1-C8, that cross react with the plasma membranes from bovine, rat and human retinal pigment epithelial cells. In radioimmunoassay both MAbD1-C6 and MAbD1-C8 had similar affinities for bovine plasma membranes. Both monoclonal antibodies identified a protein of 72 Kd with an apparent subunit of 32-35 Kd. The protein was localized to the cell surface of human and bovine retinal pigment epithelium by immunocytohistochemistry. In the normal eye the antigen identified by the monoclonal antibodies was strongly associated with the retinal pigment epithelium and weakly associated with lens tissue. Using either monoclonal antibody, components of purified bovine or rat retinal pigment epithelial plasma membranes were precipitated from solution. Based on these results, we conclude that both monoclonal antibodies are closely related and that they may be useful for the isolation and study of retinal pigment epithelial cell structure.

Animals↗

Binding of the food mutagen PhIP in pigmented tissues of mice.

The distribution of the 14C-labelled food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the tissues of C57B1/6 and NMRI mice was studied. The results showed a high and selective binding of radioactivity in the pigment epithelium of the eye and in the fur following a single dose (0.3-4 mg/kg) of [14C]PhIP in the pigmented C57B1/6 mice whereas no such localization of radioactivity was present in the albino NMRI mice. A low but selective covalent binding of radioactivity was observed in the liver, inner cortex of the kidney and in the tracheal mucosa of [14C]PhIP-injected mice. PhIP was firmly bound to synthetic melanin pigment in vitro; only 3% was released by extraction with a phosphate buffer (pH 7) whereas 72% was released by extraction by methanol:conc. NH3 (15:1). Three hours to 7 days following a single injection of [14C]PhIP in C57B1/6 mice the radioactivity in the eye was 3- to 6-fold higher than that in the liver or kidney. Almost 60% of the radioactive material present in the pigmented epithelium of the eye 3 and 24 h following injection could be extracted by basic methanol and identified as unchanged PhIP. The residual radioactivity in the pigmented epithelium of the eyes may represent a covalent binding of [14C]PhIP metabolites to cellular constituents or to a basic methanol-resistant binding of [14C]PhIP to melanin. The results indicate that pigmented tissues may be potential target tissues for the toxic effects of PhIP and suggest that the use of hair for biological monitoring of PhIP should be examined.

Animals↗