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The skin discoloration of squid subjected to frozen storage negatively affects market price. In this study, various alkali treatments were investigated for effects on red granules and yellow pigments of squid skin and corresponding mechanisms were investigated at the tissue, cellular and molecular level. A significant colour improvement was observed when subjected to a pH 12 treatment, supported by decreased Δb* and increased Δa* values. Neither lower nor harsher alkali treatments than pH 12 can not obtain such results. HE staining and the UV-vis spectrum suggest that the improved red colour in skin was ascribed to the release of red pigment granules from damaged chromatophores by alkaline treatment and the release of red pigments in alkaline aqueous solutions from granules. However, based on TEM and particle size analysis, an excessive alkali treatment of pH 13 would degrade granules into smaller particles. The degradation of yellowness pigments indicated high sensitivity to alkali environments according to HPLC results. This study provides a valuable reference for improving the colour appearance of squid skin subjected to frozen storage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cephalopods use chromatophores distributed on their soft skin to change skin color and its pattern. Each chromatophore consists of a central sac containing pigment granules and radial muscles surrounding the sac. The contraction of the radial muscle causes the central sac to expand in area, making the color of the pigment more visible. With the chromatophores actuating individually, cephalopods can create extremely complex skin color patterns, which they utilize for exquisite functions including camouflage and communication. Inspired by this mechanism, we present an artificial chromatophore that can modulate its color pattern in response to light. Multimaterial projection microstereolithography is used to integrate three functional components including a photoactive hydrogel composite with polydopamine nanoparticles (PDA-NPs), acrylic acid hydrogel, and poly(ethylene glycol) diacrylate. In order to generate light-driven actuation of the artificial chromatophore, the photothermal effect of the PDA-NPs, light-responsive deformation of the photoactive hydrogel composite, and the produced mechanical stresses are studied. Mechanical properties and interfacial bonding strengths between different materials are also investigated to ensure structural integrity during actuation. We demonstrate pattern modulation of the light-responsive artificial chromatophores (LACs) with the projection of different light patterns. The LAC may suggest a new concept for various engineering applications such as the camouflage interface, biophotonic device, and flexible display.

The predominance of the maximum at 800 nm for the light-harvesting complex LH4 (B800) and at 850 nm for LH2 (B800-850) from Rps. palustris is determined by the composition of αβ-polypeptides and pigments. In low light (LL) for Rps. palustris, strain KM 286 (1e5), along with LH4, the LL LH2 complex was synthesized with the same absorption at 800 and 850 nm. It differed from the LH4 and LH2 complex, which is synthesized under high illumination, in the composition and content of carotenoids (Car) and bacteriochlorophyll a (BChl a). LH4 differed from LL LH2 and LH2 by an additional emission maximum at 766 nm in the BChl a fluorescence spectra. All three complexes had approximately the same level (about 45%) of the energy transfer efficiency from Car to BChl a. Isolation of LL LH2 complex from Rps. palustris confirms the hypothesis of the synthesis in these bacteria under low light conditions of other types of complexes, except LH4, which is due to the multiple biosynthesis genes of αβ-polypeptides and the possibility of their various combinations.

Chromatophore organs in cephalopod skin are known to produce ultra-fast changes in appearance for camouflage and communication. Light-scattering pigment granules within chromatocytes have been presumed to be the sole source of coloration in these complex organs. We report the discovery of structural coloration emanating in precise register with expanded pigmented chromatocytes. Concurrently, using an annotated squid chromatophore proteome together with microscopy, we identify a likely biochemical component of this reflective coloration as reflectin proteins distributed in sheath cells that envelop each chromatocyte. Additionally, within the chromatocytes, where the pigment resides in nanostructured granules, we find the lens protein Ω- crystallin interfacing tightly with pigment molecules. These findings offer fresh perspectives on the intricate biophotonic interplay between pigmentary and structural coloration elements tightly co-located within the same dynamic flexible organ - a feature that may help inspire the development of new classes of engineered materials that change color and pattern.

Rapid, simple, and low-cost screening procedures are necessary for the detection of harmful compounds in the effluent that flows out of point sources such as industrial outfall. The present study investigated the effects on a novel sensor of harmful compounds such as KCN, phenol, and herbicides such as 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine (atrazine), and 2-N-tert-butyl-4-N-ethyl-6-methylsulfanyl-1,3,5-triazine-2,4-diamine (terbutryn). The sensor employed an electrode system that incorporated the photocurrent of intra-cytoplasmic membranes (so-called chromatophores) prepared from photosynthetic bacteria and linked using carbon paste electrodes. The amperometric curve (photocurrent-time curve) of photo-induced electron transfer from chromatophores of the purple photosynthetic bacterium Rhodobacter sphaeroides to the electrode via an exogenous electron acceptor was composed of two characteristic phases: an abrupt increase in current immediately after illumination (I₀), and constant current over time (Ic). Compared with other redox compounds, 2,5-dichloro-1,4-benzoquinone (DCBQ) was the most useful exogenous electron acceptor in this system. Photo-reduction of DCBQ exhibited Michaelis-Menten-like kinetics, and reduction rates were dependent on the amount of DCBQ and the photon flux intensity. The Ic decreased in the presence of KCN at concentrations over 0.05 μM (=μmol·dm(-3)). The I₀ decreased following the addition of phenol at concentrations over 20 μM. The Ic was affected by terbutryn at concentrations over 10 μM. In contrast, DCMU and atrazine had no effect on either I₀ or Ic. The utility of this electrode system for the detection of harmful compounds is discussed.

Chromatophores are pigment-bearing cells of lower vertebrates, including fish that cater for the ability of individual animals to shift body coloration and pattern. Color change provides dynamic camouflage and various kinds of communication. It is also a spectacular example of phenotypic plasticity, and of significant importance for adaptation and survival in novel environments. Through different cellular mechanisms, color change can occur within minutes or more slowly over weeks. Chromatophores have different pigment types and are located not only in the skin, but also in the eyes and internally. While morphological color change, including seasonal color change, has received a lot of interest from evolutionary biologists and behavioral ecologists, the more rapid physiological color change has been largely a research subject for cell physiologists. In this cross-disciplinary review, we have highlighted emerging trends in pigment cell research and identified unsolved problems for future research., (Copyright © 2016. Published by Elsevier Inc.)

Many chameleons, and panther chameleons in particular, have the remarkable ability to exhibit complex and rapid colour changes during social interactions such as male contests or courtship. It is generally interpreted that these changes are due to dispersion/aggregation of pigment-containing organelles within dermal chromatophores. Here, combining microscopy, photometric videography and photonic band-gap modelling, we show that chameleons shift colour through active tuning of a lattice of guanine nanocrystals within a superficial thick layer of dermal iridophores. In addition, we show that a deeper population of iridophores with larger crystals reflects a substantial proportion of sunlight especially in the near-infrared range. The organization of iridophores into two superposed layers constitutes an evolutionary novelty for chameleons, which allows some species to combine efficient camouflage with spectacular display, while potentially providing passive thermal protection.

Cephalopods can display dazzling patterns of colours by selectively contracting muscles to reversibly activate chromatophores--pigment-containing cells under their skins. Inspired by this novel colouring strategy found in nature, we design an electro-mechano-chemically responsive elastomer system that can exhibit a wide variety of fluorescent patterns under the control of electric fields. We covalently couple a stretchable elastomer with mechanochromic molecules, which emit strong fluorescent signals if sufficiently deformed. We then use electric fields to induce various patterns of large deformation on the elastomer surface, which displays versatile fluorescent patterns including lines, circles and letters on demand. Theoretical models are further constructed to predict the electrically induced fluorescent patterns and to guide the design of this class of elastomers and devices. The material and method open promising avenues for creating flexible devices in soft/wet environments that combine deformation, colorimetric and fluorescent response with topological and chemical changes in response to a single remote signal.

The chromatophores of Rhodobacter (Rb.) sphaeroides represent a minimal bio-energetic system, which efficiently converts light energy into usable chemical energy. Despite extensive studies, several issues pertaining to the morphology and molecular architecture of this elemental energy conversion system remain controversial or unknown. To tackle these issues, we combined electron microscope tomography, immuno-electron microscopy and atomic force microscopy. We found that the intracellular Rb. sphaeroides chromatophores form a continuous reticulum rather than existing as discrete vesicles. We also found that the cytochrome bc1 complex localizes to fragile chromatophore regions, which most likely constitute the tubular structures that interconnect the vesicles in the reticulum. In contrast, the peripheral light-harvesting complex 2 (LH2) is preferentially hexagonally packed within the convex vesicular regions of the membrane network. Based on these observations, we propose that the bc1 complexes are in the inter-vesicular regions and surrounded by reaction center (RC) core complexes, which in turn are bounded by arrays of peripheral antenna complexes. This arrangement affords rapid cycling of electrons between the core and bc1 complexes while maintaining efficient excitation energy transfer from LH2 domains to the RCs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Color traits in animals play crucial roles in thermoregulation, photoprotection, camouflage, and visual communication, and are amenable to objective quantification and modeling. However, the extensive variation in non-melanic pigments and structural colors in squamate reptiles has been largely disregarded. Here, we used an integrated approach to investigate the morphological basis and physical mechanisms generating variation in color traits in tropical day geckos of the genus Phelsuma., Results: Combining histology, optics, mass spectrometry, and UV and Raman spectroscopy, we found that the extensive variation in color patterns within and among Phelsuma species is generated by complex interactions between, on the one hand, chromatophores containing yellow/red pteridine pigments and, on the other hand, iridophores producing structural color by constructive interference of light with guanine nanocrystals. More specifically, we show that 1) the hue of the vivid dorsolateral skin is modulated both by variation in geometry of structural, highly ordered narrowband reflectors, and by the presence of yellow pigments, and 2) that the reflectivity of the white belly and of dorsolateral pigmentary red marks, is increased by underlying structural disorganized broadband reflectors. Most importantly, these interactions require precise colocalization of yellow and red chromatophores with different types of iridophores, characterized by ordered and disordered nanocrystals, respectively. We validated these results through numerical simulations combining pigmentary components with a multilayer interferential optical model. Finally, we show that melanophores form dark lateral patterns but do not significantly contribute to variation in blue/green or red coloration, and that changes in the pH or redox state of pigments provide yet another source of color variation in squamates., Conclusions: Precisely colocalized interacting pigmentary and structural elements generate extensive variation in lizard color patterns. Our results indicate the need to identify the developmental mechanisms responsible for the control of the size, shape, and orientation of nanocrystals, and the superposition of specific chromatophore types. This study opens up new perspectives on Phelsuma lizards as models in evolutionary developmental biology.

Chromatophores are the pigment-containing cells in the skins of animals such as fish and cephalopods which have chromomorphic (colour-changing) and controllable goniochromic (iridescent-changing) properties. These animals control the optical properties of their skins for camouflage and, it is speculated, for communication. The ability to replicate these properties in soft artificial skin structures opens up new possibilities for active camouflage, thermal regulation and active photovoltaics. This paper presents the design and implementation of soft and compliant artificial chromatophores based on the cutaneous chromatophores in fish and cephalopods. We demonstrate artificial chromatophores that are actuated by electroactive polymer artificial muscles, mimicking the radially orientated muscles found in natural chromatophores. It is shown how bio-inspired chromomorphism may be achieved using both areal expansion of dielectric elastomer structures and by the hydrostatic translocation of pigmented fluid into an artificial dermal melanophore.

Bacteriophytochromes (BphPs) are biliverdin IXα-containing photoreceptors that photoconvert between red (Pr) and far-red (Pfr) absorbing states. BphPs are one half of a two-component system that transmits a light signal to a histidine kinase domain and then to a gene-response regulator. In Rhodopseudomonas palustris, synthesis of a light-harvesting complex (LH4) is controlled by two BphPs (RpBphP2 and RpBphP3). Despite their high sequence identity (52%), their absorption spectra are very different. The spectra of RpBphP2 exhibit classic Pr-to-Pfr photoconversion, whereas RpBphP3 quenches and a high-energy Pnr state emerges [Giraud et al. (2005), J. Biol. Chem. 280, 32389-32397]. Crystallization of the chromophore-binding domain (CBD) of RpBphP2 (RpBphP2-CBD) proved to be difficult and the structure of RpBphP3-CBD was used to crystallize RpBphP2-CBD* using homologue-directed mutagenesis. The structure shows that dimerization is an important factor in successful crystallization of RpBphP2-CBD* and arises from an N136R mutation. Mutations at this site correlate with an ability to dimerize in other truncated BphPs and may also be important for full-length dimer formation. Comparison of the RpBphP3-CBD and RpBphP2-CBD* biliverdin IXα pockets revealed that the former has additional hydrogen bonding around the B and D pyrrole rings that may constrain photoconversion to Pfr, resulting in a strained photoexcited Pnr state.

Spacing variation of adjoining reflecting thin films in iridophore is responsible for the variable interference color in the paracheirodon innesi. On the basis of this phenomenon, colloidal crystal thin films with different structures are fabricated from monodisperse poly(styrene-methyl methacrylate-acrylic acid) (PSMA) colloids. The relationship between the colors and structures of the films is investigated and discussed according to the principle of light interference. A two-layer colloidal film having uniform color is researched and it displays diverse colors before and after swelling by styrene (St), which can be used to mimic the variable structural color of the paracheirodon innesi.

Bacterial photosynthetic membranes, also known as chromatophores, are tightly packed with integral membrane proteins that work together to carry out photosynthesis. Chromatophores display a wide range of cellular morphologies; spherical, tubular, and lamellar chromatophores have all been observed in different bacterial species, or with different protein constituents. Through recent computational modeling and simulation, it has been demonstrated that the light-harvesting complexes abundant in chromatophores induce local membrane curvature via multiple mechanisms. These protein complexes assemble to generate a global curvature and sculpt the chromatophores into various cellular-scale architectures.

Low-molecular-mass organogelators (LMOGs) based on photochromic molecules aggregate in selected solvents to form gels through various spatio-temporal interactions. The factors that control the mode of aggregation of the chromophoric core in the LMOGs during gelation, gelation-induced changes in fluorescence, the formation of stacked superstructures of extended pi-conjugated systems, and so forth are discussed with selected examples. Possible ways of generating various light-harvesting assemblies are proposed, and some unresolved questions, future challenges, and their possible solutions on this topic are presented.

In the photosynthetic purple bacterium Rhodobacter (Rba.) sphaeroides, light is absorbed by membrane-bound light-harvesting (LH) proteins LH1 and LH2. LH1 directly surrounds the reaction center (RC) and, together with PufX, forms a dimeric (RC-LH1-PufX)2 protein complex. In LH2-deficient Rba. sphaeroides mutants, RC-LH1-PufX dimers aggregate into tubular vesicles with a radius of approximately 250-550 A, making RC-LH1-PufX one of the few integral membrane proteins known to actively induce membrane curvature. Recently, a three-dimensional electron microscopy density map showed that the Rba. sphaeroides RC-LH1-PufX dimer exhibits a prominent bend at its dimerizing interface. To investigate the curvature properties of this highly bent protein, we employed molecular dynamics simulations to fit an all-atom structural model of the RC-LH1-PufX dimer within the electron microscopy density map. The simulations reveal how the dimer produces a membrane with high local curvature, even though the location of PufX cannot yet be determined uniquely. The resulting membrane curvature agrees well with the size of RC-LH1-PufX tubular vesicles, and demonstrates how the local curvature properties of the RC-LH1-PufX dimer propagate to form the observed long-range organization of the Rba. sphaeroides tubular vesicles.

Measuring the biological activity of hydrophobic chemicals using in vitro assays is challenging because their aqueous solubility is low and the high density of bio-suspensions strongly decreases the bioavailability of hydrophobic pollutants. Dynamic dosing by partitioning from a stable polymer has a potential to overcome these limitations. Poly(dimethylsiloxane) (PDMS) was chosen due to its documented bio-compatibility and excellent partitioning properties. PDMS sheets were loaded with five polycyclic aromatic hydrocarbons (PAHs) and then immersed in model bio-suspensions composed of membrane vesicles ("chromatophores", composed of 30% lipids and 70% proteins) isolated from the photosynthetic bacterium Rhodobacter sphaeroides or phospholipid bilayer vesicles (liposomes) composed of palmitoyl-oleoyl phosphatidylcholine (POPC). Method development included the determination of partition coefficients between chromatophores or liposomes and water, desorption rate constants from PDMS to bio-suspensions, and diffusion resistances in both PDMS and bio-suspensions. The release of the PAHs from the PDMS into the bio-suspensions was measured and modeled as a combination of diffusion in pure water and diffusion in a completely mixed solvent composed of water and bio-suspensions. The mass transfer resistance for the release was lower in the PDMS than in the tested solutions, which demonstrates that PDMS can efficiently deliver PAHs even to dense biosuspensions. The contribution of aqueous diffusion to the mass transfer decreased with increasing hydrophobicity of the PAHs indicating that hydrophobic chemicals are efficiently transported with suspended biomaterial. The passive dosing system is versatile and offers a number of applications. Promising are tests with instantaneous response, where the time-dependent effect can be translated to concentration-effect curves but the system is also applicable for assuring constant dosing for longer-term testing.

Color development of squid skin was controlled by O2 concentration for storage. When stored above 10% O2, the color index (CI) as an index of color development of skin increased in 24 h, and decreased gradually with further storage. The CI profile at 10% O2 was practically identical to that in air. When stored at 0.1% O2, in the presence of N2, the CI increased partly in 6 h and decreased. Morphological observation of chromatophore distinguished the CI increase at 0.1% and 10% O2 by their shape and size distribution. However, the storage of squid at O2 concentration between 2.5% and 7% practically did not change the CI for at least 48 h. ATP content of skin was kept unchanged when the storage atmosphere contained O2 concentration at 2.5% up to 48 h, while the content decreased rapidly with a half decrease in 6 h when stored at 0.1% O2. It was demonstrated clearly that ATP is regenerated in the presence of O2, but the ATP concentration did not determine the CI change during the storage. Exposure to high concentration of O2 might induce a full color development of squid skin.

A series of putative dipeptide substrates of prostate-specific membrane antigen (PSMA) was prepared that explored alpha- and beta/gamma-linked acidic residues at the P1 position and various chromophores at the P2 position, while keeping the P1' residue constant as L-Glu. Four chromophores were examined, including 4-phenylazobenzoyl, 1-pyrenebutyryl, 9-anthracenylcarboxyl-gamma-aminobutyryl, and 4-nitrophenylbutyryl. When evaluating these chromophores, it was found that a substrate containing 4-phenylazobenzoyl at the P2 position was consumed most efficiently. Substitution at the P1 position with acidic residues showed that only gamma-linked L-Glu and D-Glu were recognized by the enzyme, with the former being more readily proteolyzed. Lastly, binding modes of endogenous substrates and our best synthetic substrate (4-phenylazobenzoyl-Glu-gamma-Glu) were proposed by computational docking studies into an X-ray crystal structure of the PSMA extracellular domain.

A Nanodevice was constructed by delta-free F(o)F(1)-ATPase within chromatophores and actin filaments through biotinlipid-streptavidin-biotin-(AC(5))(2)Sulfo-OSu system. One actin filament linking with many chromatophores functions as the Nanodevice body and many delta-free F(o)F(1)-ATPase as the Nanodevice motors. Movement of the Nanodevice was observed directly by fluorescence microscopy with CCD camera after illumination. The moving speed was about 2.17-24.43mum/s for various length Nanodevices and most of them were stopped by adding CCCP. This means that the Nanodevice was driven by PMF (proton-motive force) in the cooperating delta-free F(o)F(1)-ATPase. From bioengineering point of view, the cooperation of F(o)F(1)-ATPase is a very important research field in the future.

Although motile iridophores in the longitudinal stripes of neon tetra skin are under control of the sympathetic nervous system, they also respond to light directly and show circadian color changes. Using neon tetra skin, we found that the photoresponse of iridophores depends on light intensity, and that light near 500 nm is most effective. RT-PCR demonstrated the expression of mRNAs encoding rhodopsin and two kinds of cone opsins (Pi-green1 and Pi-green2) in neon tetra skin where the light-sensitive iridophores exist. These mRNAs are also expressed in the lateral eyes. The cone opsin genes, Pi-green1 and Pi-green2, show high similarity with the g101 and g103 genes of unique green cone opsins (belonging to the MWS/LWS group) of the blind Mexican cavefish. These results show that Pi-green1, Pi-green2, and/or rhodopsin may play important roles in the photoresponse of neon tetra iridophores, which are most sensitive to light near 500 nm.

We present a molecular model of a chromatophore vesicle from Rhodobacter sphaeroides. These vesicles are ideal benchmark systems for molecular and systemic simulations, because they have been well studied, they are small, and they are naturally separated from their cellular environment. To set up a photosynthetic chain working under steady-state conditions, we compiled from the experimental literature the specific activities and geometries that have been determined for their constituents. This data then allowed defining the stoichiometries for all membrane proteins. This article contains the kinetic part of the reconstructed model, while the spatial reconstruction is presented in a companion article. By considering the transport properties of the Cytochrome c(2) and ubiquinone pools, we show that their size and oxidation states allow for an efficient buffering of the statistical fluctuations that arise from the small size of the vesicles. Stoichiometric and kinetic considerations indicate that a typical chromatophore vesicle of Rb. sphaeroides with a diameter of 45 nm should contain approximately five bc(1) monomers.

The photosynthetic apparatus of purple bacteria is generally considered a well-studied and understood system. However, recent atomic force microscopy images of flattened chromatophore vesicles from Rhodobacter sphaeroides restarted a debate about the stoichiometry and positions of the membrane proteins, with the interpretations of the observed images only partly being in agreement with earlier models. The most puzzling observation from the recent images is that the Cytochrome bc(1) complex, which is a central part of the photosynthetic apparatus, seems to be missing on the chromatophore vesicles, even when these were extracted from photosynthetically grown bacteria. From the available information on the geometry of the vesicle and of the proteins we reconstructed here a three-dimensional model vesicle at molecular resolution. Its central feature, also determining its diameter of approximately 45 nm, is an equatorial array of LH1 dimers, lined by a region of LH2 rings. This naturally puts the Cytochrome bc(1) complexes and the ATPase at the vesicle's poles. This spatial model may explain why the vesicle's endcaps with the bc(1) complexes are lost during the preparatory steps of the imaging process together with the ATPase and are therefore absent from the available images.

Chromatic adaptation in crustaceans results from the differential distribution of colored pigment granules within their chromatophores consequent to cell signaling by neurosecretory peptides. However, the force transducing, mechanochemical protein motors responsible for granule translocation, and their molecular mechanisms of action, are not well understood. The present study uses immunocytochemical techniques and a motility assay in vitro to demonstrate that protein motors from the kinesin and myosin superfamilies are stably associated with membrane-bounded pigment granules in the red, ovarian chromatophores of the freshwater, palaemonid shrimp, Macrobrachium olfersii. Monoclonal antibodies against conventional kinesin heavy chain, and an anti-myosin whole serum, labeled pigment-containing fragments prepared from homogenates of chromatophores with fully dispersed or aggregated pigments: this finding infers a permanent association between the protein motors and the pigment granules, and suggests that such motors may be regulated while bound to their cargos. The pigment aggregator appears to be a myosin since the anti-myosin whole serum attenuated hormonally triggered pigment aggregation in the motility assay in vitro, and induced pigment hyper-dispersion in some chromatophores. Western blots of the chromatophore-containing, ovarian tissue homogenate demonstrated protein bands consistent with myosin II and myosin XII, either of which may be the pigment aggregator. This study provides the first direct evidence for myosin and kinesin protein motors directly and stably associated with pigment granules in crustacean chromatophores, and may represent the first successful isolation of myosin class XII.

Sequence alignment of cytochrome b of the cytochrome bc1 complex from various sources reveals that bacterial cytochrome b contain an extra fragment at the C terminus. To study the role of this fragment in bacterial cytochrome bc1 complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with progressive deletion from this fragment (residues 421-445) were generated and characterized. The cytbDelta-(433-445) bc1 complex, in which 13 residues from the C-terminal end of this fragment are deleted, has electron transfer activity, subunit composition, and physical properties similar to those of the complement complex, indicating that this region of the extra fragment is not essential. In contrast, the electron transfer activity, binding of cytochrome b, ISP, and subunit IV to cytochrome c1, redox potentials of cytochromes b and c1 in the cytbDelta-(427-445), cytbDelta-(425-445), and cytbDelta-(421-445) mutant complexes, in which 19, 21, or all residues of this fragment are deleted, decrease progressively. EPR spectra of the [2Fe-2S] cluster and the cytochromes b in these three deletion mutant bc1 complexes are also altered; the extent of spectral alteration increases as this extra fragment is shortened. These results indicate that the first 12 residues (residues 421-432) from the N-terminal end of the C-terminal extra fragment of cytochrome b are essential for maintaining structural integrity of the bc1 complex.

Melanophores, xanthophores, and iridophores from the skins of the two Antarctic fish species Pagothenia borchgrevinki and Trematomus bernacchii were tested immunocytochemically for the presence of a variety of muscle proteins. Actin, myosin, and calmodulin, not surprisingly, were confirmed for all three chromatophore types of the two fishes, but the presence of caldesmon and calponin, both characteristic proteins of smooth muscle fibers, represents a new discovery. It is not known at this stage whether these proteins occur also in the chromatophores of other fishes and are not restricted to Antarctic species. Since, however, motility control of particles in fish chromatophores and the regulation of smooth muscle tension both involve the sympathetic nervous system, the presence of similar target proteins should not come as a surprise. The fact that none of the chromatophores tested positive for troponin shows that there is no close relationship between pigment cells and striated muscle. The lack of alpha-actinin in iridophores, but its presence in melanophores and xanthrophores, is thought to be a reflection of the considerably greater pigment translocations within the latter two types of chromatophore cells.

To investigate the possible photoprotective role of chromatophores in fish, the absorbances of four types of intact chromatophores in adult and larval Japanese medaka were analyzed using microspectrophotometric techniques. The absorbance spectrum of each chromatophore class was obtained from 300 to 550 nm. The absorbance spectra of intact leucophores, melanophores and xanthophores were very similar to the published absorbance spectra of the isolated pure pigments contained in each chromatophore type, pteridines, melanin and carotenoids or pteridines, respectively. Based on these absorbance spectra, leucophores and melanophores should provide the most ultraviolet (UV) photoprotection to fish since the compounds they contain, pteridines and melanin, correspondingly, have strong absorbances in the UV region of the spectrum. Xanthophores containing carotenoids are not likely to provide much protection to fish from UV-induced damage since carotenoids have low absorbances in the UV range. Xanthophores containing colored pteridines, however, may provide somewhat greater UV protection to fish, since pteridines absorb more light than carotenoids in the UV portion of the spectrum. The relative frequency, coverage and thickness of these two types of xanthophores should determine how much protection xanthophores as a chromatophore type would provide against UV-induced damage.

Bacterial chondroitinases and heparitinases are potentially useful tools for structural studies of chondroitin sulfate and heparin/heparan sulfate. Substrate specificities of Flavobacterium chondroitinase C, as well as heparitinases I and II, towards the glycosaminoglycan-protein linkage region -HexA-HexNAc-GlcA-Gal-Gal-Xyl-Ser (where HexA represents glucuronic acid or iduronic acid and HexNAc represents N-acetylgalactosamine or N-acetylglucosamine) were investigated using various structurally defined oligosaccharides or oligosaccharide-serines derived from the linkage region. In the case of oligosaccharide-serines, they were labeled with a chromophore dimethylaminoazobenzenesulfonyl chloride (DABS-Cl), which stably reacted with the amino group of the serine residue and rendered high absorbance for microanalysis. Chondroitinase C cleaved the GalNAc bond of the pentasaccharides or hexasaccharides derived from the linkage region of chondroitin sulfate chains and tolerated sulfation of the C-4 or C-6 of the GalNAc residue and C-6 of the Gal residues, as well as 2-O-phosphorylation of the Xyl residue. In contrast, it did not act on the GalNAc-GlcA linkage when attached to a 4-O-sulfated Gal residue. Heparitinase I cleaved the innermost glucosaminidic bond of the linkage region oligosaccharide-serines of heparin/heparan sulfate irrespective of substitution by uronic acid, whereas heparitinase II acted only on the glucosaminidic linkages of the repeating disaccharide region, but not on the innermost glucosaminidic linkage. These defined specificities of chondroitinase C, as well as heparitinases I and II, will be useful for preparation and structural analysis of the linkage oligosaccharides.

The pteridine content was measured as a function of age in Armadillidium vulgare, and the fine structure of the pteridine-containing granules in the integument was examined in relation to pteridine content. Yellow chromatophores are an essential component of the cream-markings, which are a defining feature of the female A. vulgare. Four kinds of pteridines in the integument including a yellow pigment (sepiapterin) were determined by HPLC. The body color of the red phenotype of A. vulgare varies from dark red to yellowish red and was formerly thought to be due to the quality and quantity of ommochrome pigment. Our analysis of the pteridine content in the integument of this phenotype revealed a significant change in sepiapterin content per body weight with age. Sepiapterin content per body weight decreased gradually with age, while that of biopterin tended to increase with age. Ultrastructural observations of the pigment granules in the yellow chromatophores revealed a corresponding change in the fine structure of pigment granules. In the older adults, some of the electron-dense fibrous materials in the pteridine-containing granules was concentrically arranged, and in the younger adults, most of pteridine-containing granules were electron-lucent. The role of pteridine quality in determining the structure of pteridine-containing granules is discussed.

We analyze the adsorption of the fluorescent monoamine 9-aminoacridine to the membrane phase of photosynthetic chromatophores, in the physiological interval of pH values ranging from 5.5 to 8.5 and at ionic strengths of 0.005 and 0.150 M. The interaction of the probe with the membrane phase is described with S-shaped isotherms of the Hill type and is modulated by electrostatic effects as modelled with the Gouy-Chapman-Boltzman theory. This description is consistent with different values of the surface change density of the chromatophore membranes decreasing from about 1.3 x 10(-3) to about 0.5 x 10(-3) e-/A2, on changing the pH from 8.5/7.5 to 6.5/5.5, respectively. Furthermore we show that, when the free concentrations of the probe in the inner and outer vesicle compartments are computed from the adsorbing isotherms at the proper pH values, the model considering the equilibrium distribution of the neutral monoamine following the onset of a delta pH is sufficient to describe the dependence of the artificially induced transmembrane delta pH values on the observed quenching of the probe fluorescence.

By whole-cell transmission electron microscopy (WCTEM), we recently demonstrated that carotenoid droplets are transported by elongating or retracting endoplasmic reticular cisternae in goldfish xanthophores. Here we report that permeabilized xanthophores demonstrate immunogold reactivity against several proteins involved in organelle translocation. The gold labeling against beta-tubulin and the intermediate filament protein p45a were found on microtubules and intermediate filaments. Labeling with anti-actin was found on nonidentifiable structures, on vesicles of unknown origin, occasional labeling on carotenoid droplets, and on occasional microfilaments. Immunoreactivity was demonstrated with anti-p57 on the carotenoid droplet surface, confirming previous results (Lynch et al., 1986a,b). Labeling with anti-PCD6 subunit (of the inositol trisphosphate/ryanodine receptor) was demonstrated on carotenoid droplets suggesting they possess calcium channels. Anti-MAP 1C (dynein) immunolabeling was generally seen on club-shaped structures in the cytomatrix and on carotenoid droplets. Finally, immunogold labeling with anti-MAP 2a + 2b was seen on a meshwork of microfilaments and intermediate filaments. Finally, this is the first report of a WCTEM technique for permeabilized cells that reveals immunoreactive elements, organelles, and cytomatrix components without the additional requirements of extraction or fracturing.

Biochemical analyses of the dorsal integument of the isopod, Armadillidium vulgare, revealed that sepiapterin, biopterin, pterin, isoxanthopterin and uric acid accumulated in the yellow-colored chromatophores which are distinguishable from ommochrome chromatophores. The pattern of the yellow-colored chromatophores in the female is externally observable at the dorsal surface of the integument as yellow markings. In contrast, the yellow-colored chromatophores are not externally observable in the male, since they are covered by an ommochrome chromatophore layer. The content of both sepiapterin and biopterin in the male chromatophores was about two times greater than that in the female. The yellow-colored chromatophores were observable by light microscopy as pigmented granules. Electron microscopy showed that morphological properties of the granules were similar to those of pteridine granules which contain uric acid occurring in the silkworm integument. These facts indicate that both pteridines and uric acid in the integument of A. vulgare are localized in the pigmented granules of the yellow-colored chromatophores.

Electron micrographs of skin from xanthic (gold) sailfin mollies revealed numerous xanthophores, as well as scattered melanophores. The melanophores were seen to contain premelanosomes in various stages of development. This is consistent with the fact that xanthic mollies have been shown to be tyrosinase positive. Melanosomes in xanthic mollies appear to develop by one of two pathways: 1) from an endoplasmic reticulum-derived vesicle which develops an internal lamellar framework, and 2) by fusion of multiple Golgi-derived vesicles which lack an internal lamellar framework. Analysis of the pigments in the skin of the xanthic mollies identified four colorless pteridine pigments (xanthopterin, isoxanthopterin, neopterin, and pterin) and a carotenoid with an absorbance spectrum similar to beta-carotene. It appears that, unlike some other poeciliid fishes, sailfin mollies do not use pteridine pigments for orange coloration. Rather, they appear to rely primarily on carotenoids.

Irides from adult Ringed Turtle Doves (Streptopelia risoria) were examined using both light and electron microscopy. The anterior surface of the iris stroma contained numerous large venous sinuses overlying bright yellow pigment cells which we classified as "reflecting xanthophores." The pigment cells were filled with irregularly arranged yellow reflecting crystals and occasional pterinosome-like structures. The irides were extracted in NaOH and the extracted pigments analyzed using paper chromatography and spectrophotometry. A unique bright orange fluorescent band was found in the iris extract, but the chemical nature of the band was not determined. Although guanine was expected to be a major component of the reflecting "platelets," based on previous work with other Columbiformes, it could not be demonstrated chromatographically or spectrophotometrically.

The stability and regulation by oxygen of mRNAs for the photosynthetic apparatus in Rhodobacter capsulatus have been studied by using proflavin to inhibit transcription and by shifting cells from anaerobic to aerobic conditions. The results from the inhibition experiments show that the mRNA for the light-harvesting LH-II polypeptides (beta, alpha) is more stable than that for the light-harvesting LH-I polypeptides (beta, alpha) during anaerobic growth, whereas the mRNAs for the reaction center polypeptides L (RC-L), M (RC-M), and H (RC-H) are less stable than both the LH-I and LH-II mRNAs. When photosynthetic cells are shifted from anaerobic to aerobic conditions, an immediate decrease in the levels of mRNA for the LH-I, LH-II, RC-L, RC-M, and RC-H proteins was observed. The level of mRNA for the LH-II proteins, however, is more sensitive to oxygen and is reduced faster than the level of mRNA for the LH-I proteins. These results suggest that oxygen represses the expression of genes coding for the light-harvesting antenna and reaction center complexes and may selectively accelerate the degradation of mRNA for the LH-II proteins. The mRNAs for several enzymes in the bacteriochlorophyll biosynthetic pathway are regulated by oxygen in a similar manner. The mRNAs for carotenoid biosynthetic enzymes, however, are regulated by oxygen in a different way. We have found that the amounts of mRNAs for carotenoid biosynthetic enzyme, relative to the amounts of mRNAs for LH and RC, increased during the shift from anaerobic to aerobic conditions. We have particularly shown that although the expression of most photosynthetic genes in R. capsulatus is repressed by oxygen, the crtA gene, located in the BamHI H fragment of the R' plasmid pRPS404 and responsible for the oxidation of spheroidene to spheroidenone, responds to oxygen in an opposite fashion. This exzymatic oxidation may protect the photosynthetic apparatus from photooxidative damage.

Melanocytes and melanin granules have been studied by electron microscopy in normal human and cat skin, and in hyperplastic human skin lesions. The melanocytes have always been found as free cells within the epidermis,i.e., on the epidermal side of the dermal membrane. Melanocytes frequently rest on the dermal membrane or bulge towards the dermis. In such cases the uninterrupted dermal membrane is uniformly thin and smooth in appearance, in contrast with the regions alongside Malpighian cells, where it appears appreciably thicker and seemingly anchored to the basal cell layer. Two types of melanin granules have been distinguished according to their location in the melanocytes and to morphological characteristics which may only express different stages in the maturation of the granules: (a) light melanin granules in which a structure resembling a fine network is apparent; (b) dense melanin granules which, in osmium-fixed preparations, appear as uniformly dense masses surrounded by a coarsely granular, intensely osmiophilic shell. Treatment of sections of osmium-fixed tissues with potassium permanganate has revealed within the dense granules the existence of an organized framework in the form of a regular, crystalline-like lattice. It is suggested that this basic structure is protein in nature and may include the enzymatic system capable of producing melanin. The existence is reported of fine filaments located in the cytoplasm of melanocytes and morphologically distinct from the tonofilaments found in Malpighian cells.