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PubMed1994

What makes red visual pigments red? A resonance Raman microprobe study of retinal chromophore structure in iodopsin.

We have obtained resonance Raman spectra of iodopsin, a red-sensitive (lambda max 571 nm) pigment from chicken cone cells, to investigate the molecular mechanism of the opsin shift in visual pigments. Detergent-solubilized iodopsin samples were examined with a Raman microprobe to obtain spectra from a 77-K photostationary steady-state mixture composed of 11-cis-iodopsin and its 9-cis-isoiodopsin and all-trans-bathoiodopsin photoproducts. The vibrational modes of these species have been assigned by comparison with spectra of the corresponding bovine pigments. The single bond stretching frequencies of the bovine, toad, and chicken pigments are found to exhibit a regular correlation as a function of the pigment absorption maxima that is consistent with the expected effects of increased electron delocalization. The C = NH stretching frequencies of iodopsin and bathoiodopsin are at 1644 and 1638 cm-1, respectively, and shift down to 1621 and 1617 cm-1, respectively, when the nitrogen is deuterated. The C = ND stretching frequencies of the various pigments are found to decrease linearly with increasing absorption maxima, suggesting that at least part of the opsin shift in visual pigments results from weakened electrostatic interaction between the retinal chromophore and its protein counterion. The Raman data are inconsistent with the idea that a charged protein residue is shifted along the chromophore to regulate the opsin shift. Taken together with the mutagenesis and model compound results, these resonance Raman data suggest that the opsin shift between the green and red cone visual pigment arises from two effects. First, Tyr-274 provides increased electrostatic stabilization of the Schiff base-counterion ion pair. Second, the opsin shift is enhanced by the dipolar residues Ser-177 and Thr-282 that interact with the chromophore near the ionone ring to preferentially stabilize the highly dipolar charge distribution of the electronically excited retinal chromophore [Mathies, R., & Stryer, L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2169-2173].

Animals↗
PubMed2003

Molecular basis of spectral tuning in the newt short wavelength sensitive visual pigment.

Previously we reported the sequence of the member of the short wavelength sensitive 2 (SWS2) family of vertebrate visual pigments from the retina of the Japanese common newt, Cynops pyrrhogaster[Takahashi, Y. et al. (2001) FEBS Lett. 501, 151-155]. Now we have expressed the apopigment and regenerated it with A1 retinal. Its absorption maximum, 474 nm, is greatly red shifted compared to other known SWS2 pigments (418-455 nm). To determine the amino acid residues that control its spectral tuning, we replaced the residues that were near the chromophore and which differed between the newt and the bullfrog (lambda(max) = 430 nm) wild-type SWS2 pigments: Pro91Ser, Ser94Ala, Ile122Met, Cys127Ser, Ser211Cys, Tyr261Phe, and Ala292Ser. Each of these site-directed mutants led to blue shifts of the newt pigment with five of them causing substantial shifts; their sum was about equal to the difference between the absorption maximum of the bullfrog and newt pigments, 44 nm. The 32 nm shift of the absorption maximum of the multiple seven-residue mutant to 442 nm is fairly close to that of the wild-type bullfrog pigment. Thus, the seven amino acid residues that we replaced are the major cause of the red shift of the newt SWS2 pigment's spectrum. Two of the residues, 91 and 94, have not previously been identified as wavelength regulating sites in visual pigments. One of these, 91, probably regulates color via a new mechanism: altering of a hydrogen bonding network that is connected via a water to the chromophore, in this case its counterion, Glu113.

Amino Acid Sequence↗
PubMed2003

Color characteristics of monascus pigments derived by fermentation with various amino acids.

Various pigment colors were produced by Monascus fermentations with separate addition of 20 amino acids. The color characteristics and structures of the pigment derivatives were investigated. When each amino acid was added to the fermentation broth as a precursor, pigment extracts with different hue and chroma values were obtained depending on the content ratios of yellow, orange, and red colors in the fermentation broth. The yellow and orange pigments were identical regardless of amino acid addition. The red compounds varied on the basis of the type of amino acid added. LC-MS and (1)H and (13)C NMR structural analyses confirmed that the derivative pigments contained the moieties of the added amino acids. L, a, and b values of the CIELAB color system for the derivative pigments were measured. Values of hue and chroma were then calculated. The colors of the derivative pigments were in the range of orangish red to violet red. The hydrophilicities/hydrophobicities of the derivative pigments could be predicted from their log P values, which were estimated using computer programs.

Amino Acids↗
PubMed2003

Characterization of dark liver pigment observed in rats after subchronic dosing of the beta3-adrenergic receptor agonist LY368842.

Dark liver pigmentation was observed in F344 rats in a subchronic toxicology study after daily dosing of LY368842 glycolate. In addition, green-colored urine was observed in some animals. To identify the source of the pigment and its potential for toxic consequences, the liver pigment was isolated from the liver tissue of rats. The resulting material was a dark brown to black powder that was insoluble in water, organic solvents, or a tissue-solubilizing agent. Several techniques, such as chemical degradation, HPLC, tandem mass spectrometry (LC/MS/MS), (1)H NMR, and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), were employed to characterize the dark liver pigment. Following oxidative degradation of the isolated pigment, degradation products related to LY368842 were identified or tentatively identified using LC/MS/MS. Two degradation products had the same protonated molecular ion at m/z 505, which is 30 amu higher than that of LY368842. The major m/z 505 product has been identified as the indole-2,3-dione oxidative product based on (1)H NMR data and confirmed by an authentic standard. In addition, monohydroxylated product was also identified in the degradation mixture. These degradation products were consistent with the metabolites found in vivo in rats. MALDI-MS analyses of liver and urine pigment both identified a product with a protonated molecular ion at m/z 977, suggesting formation of indirubin-like and indigo-like pigments. The results obtained suggest that the oxidative metabolites of LY368842 played a key role in the formation of the liver and urine pigments.

Adrenergic beta-3 Receptor Agonists↗
PubMed2000

Melanosome capping of keratinocytes in pigmented reconstructed epidermis--effect of ultraviolet radiation and 3-isobutyl-1-methyl-xanthine on melanogenesis.

Reconstructed pigmented epidermis was established by co-seeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air-liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3-isobutyl-1-methyl-xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus. In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity. In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied.

1-Methyl-3-isobutylxanthine↗
PubMed2003

Diversity of visual pigments from the viewpoint of G protein activation--comparison with other G protein-coupled receptors.

The visual pigment present in the photoreceptor cells of the retina is a member of the family of G protein-coupled receptors and contains an 11-cis-retinal as a light-absorbing chromophore. Light induces conformational changes in the protein moiety of the visual pigment through cis-trans isomerization of the chromophore, which leads to the activation of a G protein-mediated signal transduction cascade that eventually generates an electrical response of the photoreceptor cells. So far, various types of visual pigments have been identified from a variety of photoreceptor cells and the structure-function relationship of visual pigments has been widely investigated by means of biophysical, biochemical and molecular biological techniques. Recent identifications of visual pigment-like proteins in the extra-ocular cells emphasize the importance of the visual pigment family as the photoreceptive molecules in not only visual but also non-visual photoreception. This article reviews the functional diversity of visual pigments from the viewpoint of the molecular mechanisms of photoreception and G protein activation. In addition, the similarity and difference of G protein activation mechanism between visual pigment and other G protein-coupled receptors are discussed for furthering our understanding of the common mechanism of G protein activation by G protein-coupled receptors.

Animals↗
PubMed2002

Histamine is involved in ultraviolet B-induced pigmentation of guinea pig skin.

We previously reported that histamine induced melanogenesis in cultured human melanocytes and that the stimulatory effect was mediated by protein kinase A activation via H2 receptors. It is well-known that ultraviolet B irradiation causes acute inflammation, known as erythema, and subsequent pigmentation, and there are several reports demonstrating an elevation of the histamine levels in ultraviolet B-irradiated skin. Thus, to evaluate the involvement of histamine in ultraviolet B-induced skin pigmentation, we examined the effect of an H2 antagonist in brownish guinea pig skin. Daily exposure to 200 mJ per cm2 ultraviolet B for 3 d evoked erythema and subsequent pigmentation in the skin samples tested. Moreover, a remarkable increase in dopa-positive melanocytes was observed in the pigmented area, which showed an increase in melanin synthesis. Topical application of famotidine, an H2 antagonist, significantly reduced pigmentation and moderated the increase of dopa-positive melanocytes in the ultraviolet B-irradiated skin. Even when the initiation of famotidine application was delayed to day 2 after irradiation, an inhibitory activity on ultraviolet B-induced pigmentation was observed; however, the ultraviolet B-induced erythema was not suppressed by topically applied famotidine. Thus, we concluded that histamine is involved in ultraviolet B-induced pigmentation and that famotidine suppressed the pigmentation by the prevention of histamine binding to H2 receptors in melanocytes but not by prevention of ultraviolet B permeability and inflammation.

Animals↗
PubMed1998

Facultative skin pigmentation in caucasians: an objective biological indicator of lifetime exposure to ultraviolet radiation?

To investigate age and gender trends in facultative and constitutive skin pigmentation we measured skin pigmentation non-invasively and objectively by skin reflectance spectroscopy in 653 caucasians (336 females and 317 males; mean age 38 years, range 0-85) who were not using artificial tanning devices. In all subjects, measurements were performed in the late winter and pre-spring period at five sites exposed to ultraviolet (UV) radiation: the forehead, the upper chest, the upper back and the lateral and medial aspects of the upper arm, and in UV-unexposed buttock skin. Constitutive pigmentation at the buttocks was highest in the first years of life and then decreased substantially during the first two decades of life (P < 0.01). After the age of 25 years, buttock pigmentation remained at a constant level (P = 0.20). There was no gender difference in constitutive pigmentation. Facultative skin pigmentation increased with age for all the measured sites with the highest levels found at the lateral aspect of the upper arm. Based on observations in this study we propose the idea of a 'sun exposure index' (SEI) for individuals, based on objective measurements of skin pigmentation. The SEI is calculated as the increase in facultative pigmentation above the constitutive level and is expressed as a percentage of the constitutive level. The SEI appeared to be related to cumulative lifetime UV exposure and may be used in epidemiological research as an objective estimate of UV exposure at different body sites in caucasians.

Adolescent↗
PubMed2001

Pigmentation in Koreans: study of the differences from caucasians in age, gender and seasonal variations.

BACKGROUND: Human skin colour shows variations throughout life, and many extrinsic and intrinsic factors influence melanogenesis. Facultative pigmentation of sun-exposed skin has been suggested to reflect cumulative lifetime ultraviolet (UV) exposure in caucasians. However, pigmentary changes due to various regulatory factors may be different in dark-skinned peoples. OBJECTIVES: To observe the variations in skin colour due to ageing, gender differences and seasonal changes in Koreans with skin type IV or V. METHODS: Skin pigmentation was measured at five body sites (buttock, glabella, the V of the neck area, inner arm and dorsal forearm) using skin reflectance spectroscopy in 497 subjects (age range 0-87 years) in winter and 311 subjects (age range 0-84 years) in summer. Among these subjects, 110 were assessed in both seasons. Three independent measurements at each site were done and the average value was used as the pigmentation level. RESULTS: Constitutive pigmentation of the buttock was highest in the first decade of life. It then decreased during the second decade and this decreased level was maintained after the third decade. In contrast to caucasians, facultative pigmentation and sun exposure index did not increase with ageing. Gender differences were significant at all body sites after the first decade. Seasonal changes were apparent in dorsal forearm pigmentation. Little difference was seen in forehead pigmentation between summer and winter. CONCLUSIONS: Basal melanogenic regulation might not be different between Asians and caucasians. However, the sun exposure index may not represent lifelong cumulative UV exposure in Koreans. Age-, gender- and season-related characteristics of skin pigmentation in Koreans imply that genetically determined basal skin colour plays an important part in characterizing later responsiveness to UV radiation and sex hormones. Understanding differences between races will be helpful in studying the regulatory mechanisms of melanogenesis.

Adolescent↗
PubMed2000

Functional expression of a locust visual pigment in transgenic Drosophila melanogaster.

The cDNA encoding a visual pigment of the locust Schistocerca gregaria has been inserted into the germline of the ninaE mutant of Drosophila melanogaster by P-element-mediated transformation. Functional expression has been documented by recording light-regulated electroretinograms in transgenic flies. The spectral properties of the expressed visual pigment were determined with detergent-solubilized material, prepared from the eyecups of the transgenic D. melanogaster. The recombinant locust pigment, as well as the genuine pigment of the fruitfly (Rh1) that served as a control for transformation/expression, showed photoreversibility between the pigment and metapigment forms. The absorptions of the difference spectra identify the locust visual pigment as a short wavelength-absorbing, blue-light-sensitive photoreceptor. The absorption maxima are similar to those recorded on living locust animals. These results show that, although locust visual pigments contain 11-cis retinal as chromophore, the expressed protein is able to adopt 3-hydroxyretinal that is provided by the transgenic fruitflies. The electrophysiological recordings reveal that the locust visual pigment is able to induce phototransduction in the fruitfly. The reported results have two important consequences: On the one hand, the binding site of the locust opsin is apparently able to interact with the 3-hydroxyretinal from Drosophila in a way that the biological signal generated by the photoisomerization of the chromophore can be used by the protein to adopt a physiologically active conformation. On the other hand, despite the relatively large phylogenetic distance between both insect species, the extent of conservation between the protein domains thought to be involved in G-protein activation is striking.

Animals↗
PubMed2002

Correlation between pigmentation and antifouling compounds produced by Pseudoalteromonas tunicata.

Pseudoalteromonas tunicata is a marine bacterium with the ability to prevent biofouling by the production of at least four target-specific compounds. In addition to these antifouling compounds, P. tunicata produces at least two pigments. These include a yellow and a purple pigment which, when combined, give the bacterium a dark green appearance. Transposon mutagenesis was used in this study to investigate the correlation between pigment production and the expression of specific antifouling phenotypes in P. tunicata. Four different categories of pigmentation mutants were isolated including yellow, dark-purple, light-purple and white mutants. The mutants were tested for their ability to inhibit the settlement of invertebrate larvae, algal spore germination, fungal growth and bacterial growth. The results showed that the yellow-pigmented mutants retained full antifouling activity, whereas the purple and white mutant strains had lost some, or all, of their ability to inhibit target organisms. This demonstrates that the loss of antifouling capabilities correlates with the loss of yellow pigment and not purple pigment. Sequencing and analysis of the genes disrupted by the transposons in these mutants identified a number of potential biosynthetic enzymes and transport systems involved in the synthesis and regulation of pigmentation and fouling inhibitors in this organism.

DNA Transposable Elements↗
PubMed2003

Influence of hydration on dihydroxyacetone-induced pigmentation of stratum corneum.

Dihydroxyacetone, the browning ingredient in sunless tanning formulations, reacts with amino acids in the outer stratum corneum to form a mixture of high molecular weight pigments. Our initial observations indicated that high hydration of dihydroxyacetone-treated skin completely inhibited development of pigmentation. To investigate the mechanism underlying this effect, studies were carried out in isolated murine epidermis, polyvinyl alcohol/lysine films, and lysine in glycerol/water solvent. Murine epidermis treated with dihydroxyacetone showed a biphasic dependence on relative humidity: maximum pigmentation developed at 84% relative humidity and minimum pigmentation at 0% and 100% relative humidity. Filaggrin proteolysis, which shows a similar dependence on relative humidity and provides free amino acids in the outer stratum corneum, did not account for the relative humidity dependence of dihydroxyacetone pigmentation. A similar biphasic pigmentation response was obtained when polyvinyl alcohol film containing lysine was treated with dihydroxyacetone and incubated at various relative humidities, indicating that the structure of the stratum corneum was not a major factor. To remove the influence of the matrix, the reaction of dihydroxyacetone with lysine was followed at varying concentrations of water in mixed glycerol/buffer solvent. Again, greater pigment formation was found at an intermediate level of water (6% vol/vol) and little pigmentation at 0% and 100% water content. These results are consistent with a requirement for water at low relative humidity, which facilitates formation of free amine groups needed for the initial reaction with dihydroxyacetone, and with inhibition of the dehydration reactions by water through the law of mass action at high relative humidity.

Amino Acids↗
PubMed2003

Pigmented eccrine poroma: a report of two cases and study with dermatoscopy.

BACKGROUND: Most eccrine poromas are devoid of melanin pigmentation clinically and melanocytes on microscopic examination. Pigmented variant of eccrine poroma has been reported only sporadically. Dermatoscopy (epiluminescence microscopy) has recently proved to be a useful, noninvasive technique for the diagnosis of cutaneous pigmented lesions. However, detailed information about the pigmented variant of eccrine poroma is not available. METHOD: We report two additional cases of pigmented eccrine poroma and describe their dermatoscopic features. RESULT: The dermatoscopic features of pigmented poromas except the maple leaf-like structures and spoke-wheel areas were similar to those of pigmented basal cell carcinomas. CONCLUSION: Physicians therefore should be aware of the dermatoscopic differentiation between pigmented poromas and basal cell carcinomas.

Acrospiroma↗
PubMed2001

[Dependence of lipid peroxidation on pigmentation of the porcine iris].

PURPOSE: Melanin has been shown to act as antioxidant in lipid peroxidation studies. We have now investigated lipid peroxidation in dependence on stromal pigmentation in isolated porcine irises. METHODS: The same number of lightly pigmented and heavily pigmented porcine irises (visual selection) were homogenized in buffer (50 mmol/l Na2HPO4, 50 mmol/l NaH2PO4 and 4 mmol/l sodium azide; 1:20 w/v). 500 microliters homogenate were incubated at 37 degrees C for 5, 10, 20 and 40 min in absence and presence of Fe2+ as inducer of lipid peroxidation. Lipid peroxidation was assayed by the thiobarbituric acid (TBA) test. Results are expressed as nmol of TBA reactive material produced (TBAR) per mg protein. Fe2+ concentration of the supernatant was determined spectrophotometrically with phenanthroline. RESULTS: 70 mumol/l, 180 mumol/l and 360 mumol/l Fe2+ induced lipid peroxidation. A plateau region was reached after 20 min. Lipid peroxidation differed in dependence on stromal pigmentation in porcine irises by a factor of 2.8. 180 mumol/l Fe2+ induced 1.373 +/- 0.138 nmol TBAR/mg protein in lightly pigmented irises compared to 0.491 +/- 0.125 nmol TBAR/mg protein in heavily pigmented irises after 10 min incubation (p < 0.0001, n = 4). On the other hand, the content of Fe2+ in the supernatant was the same within error. CONCLUSIONS: There was a stronger induction of lipid peroxidation in lightly pigmented porcine irises compared to heavily pigmented porcine irises. This effect may be related to the difference in stromal melanin content and its antioxidant activity.

Animals↗
PubMed1989

Why are blue visual pigments blue? A resonance Raman microprobe study.

A resonance Raman microscope has been developed to study the structure of the retinal prosthetic group in the visual pigments of individual photoreceptor cells. Raman vibrational spectra are obtained by focusing the probe laser on intact photoreceptors frozen on a 77 K cold stage. To elucidate the mechanism of wavelength regulation in blue visual pigments, we have used this apparatus to study the structure of the chromophore in the 440-nm absorbing pigment found in "green rods" of the toad (Bufo marinus). The 9-cis isorhodopsin form of the green rod pigment exhibits a 1662-cm-1 C = NH+ Schiff base stretching mode that shifts to 1636 cm-1 in deuterium-substituted H2O. This demonstrates that the Schiff base linkage to the protein is protonated. Protonation of the Schiff base is sufficient to explain the 440-nm absorption maximum of this pigment without invoking any additional protein-chromophore interactions. The absence of additional perturbations is supported by the observation that the ethylenic band and the perturbation-sensitive C-10-C-11 and C-14-C-15 stretching modes have the same frequency as those of the 9-cis protonated retinal Schiff base in solution. Our demonstration that a blue visual pigment contains an unperturbed protonated Schiff base provides experimental evidence that the protein charge perturbation responsible for the opsin shift in the 500-nm absorbing pigments is removed in the opsins of blue pigments, as suggested by the sequence data.

Animals↗
PubMed1999

Mutually exclusive expression of human red and green visual pigment-reporter transgenes occurs at high frequency in murine cone photoreceptors.

This study examines the mechanism of mutually exclusive expression of the human X-linked red and green visual pigment genes in their respective cone photoreceptors by asking whether this expression pattern can be produced in a mammal that normally carries only a single X-linked visual pigment gene. To address this question, we generated transgenic mice that carry a single copy of a minimal human X chromosome visual pigment gene array in which the red and green pigment gene transcription units were replaced, respectively, by alkaline phosphatase and beta-galactosidase reporters. As determined by histochemical staining, the reporters are expressed exclusively in cone photoreceptor cells. In 20 transgenic mice carrying any one of three independent transgene insertion events, an average of 63% of expressing cones have alkaline phosphatase activity, 10% have beta-galactosidase activity, and 27% have activity for both reporters. Thus, mutually exclusive expression of red and green pigment transgenes can be achieved in a large fraction of cones in a dichromat mammal, suggesting a facile evolutionary path for the development of trichromacy after visual pigment gene duplication. These observations are consistent with a model of visual pigment expression in which stochastic pairing occurs between a locus control region and either the red or the green pigment gene promotor.

Animals↗
PubMed1994

Intracellular calcium and cAMP regulate directional pigment movements in teleost erythrophores.

Teleost pigment cells (erythrophores and melanophores) are useful models for studying the regulation of rapid, microtubule-dependent organelle transport. Previous studies suggest that melanophores regulate the direction of pigment movements via changes in intracellular cAMP (Rozdzial and Haimo, 1986a; Sammak et al., 1992), whereas erythrophores may use calcium- (Ca(2+)-) based regulation (Luby-Phelps and Porter, 1982; McNiven and Ward, 1988). Despite these observations, there have been no direct measurements in intact erythrophores or any cell type correlating changes of intracellular free Ca2+ ([Ca2+]i) with organelle movements. Here we demonstrate that extracellular Ca2+ is necessary and that a Ca2+ influx via microinjection is sufficient to induce pigment aggregation in erythrophores, but not melanophores of squirrel fish. Using the Ca(2+)-sensitive indicator, Fura-2, we demonstrate that [Ca2+]i rises dramatically concomitant with aggregation of pigment granules in erythrophores, but not melanophores. In addition, we find that an erythrophore stimulated to aggregate pigment will immediately transmit a rise in [Ca2+]i to neighboring cells, suggesting that these cells are electrically coupled. Surprisingly, we find that a fall in [Ca2+]i is not sufficient to induce pigment dispersion in erythrophores, contrary to the findings obtained with the ionophore and lysed-cell models (Luby-Phelps and Porter, 1982; McNiven and Ward, 1988). We find that a rise in intracellular cAMP ([cAMP]i) induces pigment dispersion, and that this dispersive stimulus can be overridden by an aggregation stimulus, suggesting that both high [cAMP]i and low [Ca2+]i are necessary to produce pigment dispersion in erythrophores.

Animals↗
PubMed1977

Transformations in the structure of the cytoplasmic ground substance in erythrophores during pigment aggregation and dispersion. I. A study using whole-cell preparations in stereo high voltage electron microscopy.

Pigment migration in cultured erythrophores of the squirrel fish Holocentrus ascensionis, after manipulation with K+, epinephrine, 3',5'-dibutyryl cyclic adenosine monophosphate, theophylline, and caffeine, is essentially identical to that observed in this chromatophore in situ. For such observations, the erythrophores are dissociated from the scales with hyaluronidase and collagenase, and allowed to spread on an amorphous collagen substrate, where they resemble the discoid erythrophore in situ. In this state, they are readily fixed by glutaraldehyde and osmium tetroxide, and are then critical-point dried for whole-cell viewing in the high voltage electron microscope. The organization and fine structure of the erythrophore cytoplast was stereoscopically examined after fixation of the pigment granules in four experimental states: pigment dispersed, pigment aggregated, pigment aggregating, and pigment dispersing. In the dispersed cell, granules are contained in an extensive three-dimensional lattice composed of radially oriented microtubules and a network of fine filaments 3-6 nm in diameter (microtrabeculae), whereas in the aggregated cell, the microtrabecular system is absent, and the majority of the microtubules appear displaced into the cortices on the cytoplasmic surface of the plasma membrane. In cells fixed while aggregating, few microtrabeculae are observed, although formless thickenings are observed in the cortices, on granules, and between clumped granules. In dispersing cells, the microtrabecular system is reformed from material stored in the cortices and with the granules in the centrosphere. These observations suggest that the granules are suspended in a dynamic microtrabecular system that withdraws during pigment aggregation and is restructured during pigment dispersion. The microtubules guide linear granule motion not by defining physical channels, but by a recognizable affinity of microtubules, microtrabeculae, and granules for one another.

Animals↗