Substrate specificity studies of Flavobacterium chondroitinase C and heparitinases towards the glycosaminoglycan--protein linkage region. Use of a sensitive analytical method developed by chromophore-labeling of linkage glycoserines using dimethylaminoazobenzenesulfonyl chloride. (1/112)

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.  (+info)

Stripe formation in juvenile Pomacanthus explained by a generalized turing mechanism with chemotaxis. (2/112)

Current interest in pattern formation can be traced to a seminal paper by Turing, who demonstrated that a system of reacting and diffusing chemicals, called morphogens, can interact so as to produce stable nonuniform concentration patterns in space. Recently, a Turing model has been suggested to explain the development of pigmentation patterns on species of growing angelfish such as Pomacanthus semicirculatus, which exhibit readily observed changes in the number, size, and orientation of colored stripes during development of juvenile and adult stages, but the model fails to predict key features of the observations on stripe formation. Here we develop a generalized Turing model incorporating cell growth and movement, we analyze the effects of these processes on patterning, and we demonstrate that the model can explain important features of pattern formation in a growing system such as Pomacanthus. The applicability of classical Turing models to biological pattern formation is limited by virtue of the sensitivity of patterns to model parameters, but here we show that the incorporation of growth results in robustly generated patterns without strict parameter control. In the model, chemotaxis in response to gradients in a morphogen distribution leads to aggregation of one type of pigment cell into a striped spatial pattern.  (+info)

Hybrid Rhodospirillum rubrum F(0)F(1) ATP synthases containing spinach chloroplast F(1) beta or alpha and beta subunits reveal the essential role of the alpha subunit in ATP synthesis and tentoxin sensitivity. (3/112)

Trace amounts ( approximately 5%) of the chloroplast alpha subunit were found to be absolutely required for effective restoration of catalytic function to LiCl-treated chromatophores of Rhodospirillum rubrum with the chloroplast beta subunit (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072). To clarify the role of the alpha subunit in the rebinding of beta, restoration of catalytic function, and conferral of sensitivity to the chloroplast-specific inhibitor tentoxin, LiCl-treated chromatophores were analyzed by immunoblotting before and after reconstitution with mixtures of R. rubrum and chloroplast alpha and beta subunits. The treated chromatophores were found to have lost, in addition to most of their beta subunits, approximately a third of the alpha subunits, and restoration of catalytic activity required rebinding of both subunits. The hybrid reconstituted with the R. rubrum alpha and chloroplast beta subunits was active in ATP synthesis as well as hydrolysis, and both activities were completely resistant to tentoxin. In contrast, a hybrid reconstituted with both chloroplast alpha and beta subunits restored only a MgATPase activity, which was fully inhibited by tentoxin. These results indicate that all three copies of the R. rubrum alpha subunit are required for proton-coupled ATP synthesis, whereas for conferral of tentoxin sensitivity at least one copy of the chloroplast alpha subunit is required together with the chloroplast beta subunit. The hybrid system was further used to examine the effects of amino acid substitution at position 83 of the beta subunit on sensitivity to tentoxin.  (+info)

The calcium dependence of pigment translocation in freshwater shrimp red ovarian chromatophores. (4/112)

The roles of calcium in cell signaling consequent to chromatophorotropin action and as an activator of mechanochemical transport proteins responsible for pigment granule translocation were investigated in the red ovarian chromatosomes of the freshwater shrimp Macrobrachium olfersii. Chromatosomes were perfused with known concentrations of free Ca++ (10(-3) to 10(-9) M) prepared in Mg(++)-EGTA-buffered physiological saline after selectively permeabilizing with 25 microM calcium ionophore A23187 or with 10(-8) M red pigment concentrating hormone (RPCH). The degree of pigment aggregation and the translocation velocity of the leading edges of the pigment mass were recorded in individual chromatosomes during aggregation induced by RPCH or A23187 and dispersion induced by low Ca++. Aggregation is Ca++ dependent, showing a dual extracellular and intracellular requirement. After perfusion with reduced Ca++ (10(-4) to 10(-9) M), RPCH triggers partial aggregation (approximately 65%), although the maximum translocation velocities (approximately 16.5 microns/min) and velocity profiles are unaffected. After aggregation induced at or below 10(-5) M Ca++, spontaneous pigment dispersion ensues, suggesting a Ca++ requirement for RPCH coupling to its receptor, or a concentration-dependent, Ca(++)-induced Ca(++)-release mechanism. The Ca(++)-channel blockers Mn++ (5 mM) and verapamil (50 microM) have no effect on RPCH-triggered aggregation. An intracellular Ca++ requirement for aggregation was demonstrated in chromatosomes in which the Ca++ gradient across the cell membrane was dissipated with A23187. At free [Ca++] above 10(-3) M, aggregation is complete; at 10(-4) M, aggregation is partial, followed by spontaneous dispersion; below 10(-5) M Ca++, pigments do not aggregate but disperse slightly. Aggregation velocities diminish from 11.6 +/- 1.2 microns/min at 5.5 mM Ca++ to 7.4 +/- 1.3 microns/min at 10(-4) M Ca++. Half-maximum aggregation occurs at 3.2 x 10(-5) M Ca++ and half-maximum translocation velocity at 4.8 x 10(-5) M Ca++. Pigment redispersion after 5.5 mM Ca(++)-A23187-induced aggregation is initiated by reducing extracellular Ca++: slight dispersion begins at 10(-7) M, complete dispersion being attained at 10(-9) M Ca++. Dispersion velocities increase from 0.6 +/- 0.2 to 3.1 +/- 0.5 microns/min. Half-maximum dispersion occurs at 7.6 x 10(-9) M Ca++ and half-maximum translocation velocity at 2.9 x 10(-9) M Ca++. These data reveal an extracellular and an intracellular Ca++ requirement for RPCH action, and demonstrate that the centripetal or centrifugal direction of pigment movement, the translocation velocity, and the degree of pigment aggregation or dispersion attained are calcium-dependent properties of the granule translocation apparatus.  (+info)

Reflective properties of iridophores and fluorescent 'eyespots' in the loliginid squid Alloteuthis subulata and Loligo vulgaris. (5/112)

Observations were made of the reflective properties of the iridophore stripes of the squid Alloteuthis subulata and Loligo vulgaris, and the likely functions of these stripes are considered in terms of concealment and signalling. In both species, the mantle muscle is almost transparent. Stripes of iridophores run along the length of each side of the mantle, some of which, when viewed at normal incidence in white light, reflect red, others green or blue. When viewed obliquely, the wavebands best reflected move towards the blue/ultraviolet end of the spectrum and their reflections are almost 100% polarised. These are properties of quarter-wavelength stacks of chitin and cytoplasm, predicted in theoretical analyses made by Sir A. F. Huxley and Professor M. F. Land. The reflecting surfaces of the individual iridophores are almost flat and, in a given stripe, these surfaces are within a few degrees of being parallel. Both species of squid have conspicuous, brightly coloured reflectors above their eyes. These 'eyespots' have iridescent layers similar to those found on the mantle but are overlaid by a green fluorescent layer that does not change colour or become polarised as it is viewed more obliquely. In the sea, all reflections from the iridophore stripes will be largely confined to the blue-green parts of the spectrum and all reflections in other wavebands, such as those in the red and near ultraviolet, will be weak. The functions of the iridophores reflecting red at normal incidence must be sought in their reflections of blue-green at oblique angles of incidence. These squid rely for their camouflage mainly on their transparency, and the ventral iridophores and the red, green and blue reflective stripes must be used mainly for signalling. The reflectivities of some of these stripes are relatively low, allowing a large fraction of the incident light to be transmitted into the mantle cavity. Despite their low reflectivities, the stripes are very conspicuous when viewed from some limited directions because they reflect light from directions for which the radiances are much higher than those of the backgrounds against which they are viewed. The reflective patterns seen, for example, by neighbouring squid when schooling depend on the orientation of the squid in the external light field and the position of the squid relative to these neighbours.  (+info)

Biochemical characterization of crystals from the dermal iridophores of a chameleon Anolis carolinensis. (6/112)

The biochemical characteristics of dermal iridophore crystals from Anolis carolinensis have been investigated. Iridophores isolated by collangenase-hyaluronidase treatment were sonicated and their contents fractionated through sucrose. Pure iridophore crystals so obtained were examined by chromatography and electron diffraction. They were found to be pure hydrated crystalline form. The suggestion is made that the subcrystalline structure of this guanine does not play a role in color production by the iridophore.  (+info)

Behavioral visual responses of wild-type and hypopigmented zebrafish. (7/112)

Zebrafish possess three classes of chromatophores that include iridophores, melanophores, and xanthophores. Mutations that lack one or two classes of chromatophores have been isolated or genetically constructed. Using a behavioral assay based on visually mediated escape responses, we measured the visual response of fully and partially pigmented zebrafish. In zebrafish that lack iridophores (roy mutants), the behavioral visual responses were similar to those of wild-type animals except at low contrast stimulation. In the absence of melanophores (albino mutants) or both melanophores and iridophores (ruby mutants), the behavioral visual responses were normal under moderate illumination but reduced when tested under dim or bright conditions or under low contrast stimulation. Together, the data suggest that screening pigments in the retina play a role in the regulation of behavioral visual responses and are necessary for avoiding "scatter" under bright light conditions.  (+info)

Temporal and cellular requirements for Fms signaling during zebrafish adult pigment pattern development. (8/112)

Ectothermic vertebrates exhibit a diverse array of adult pigment patterns. A common element of these patterns is alternating dark and light stripes each comprising different classes of neural crest-derived pigment cells. In the zebrafish, Danio rerio, alternating horizontal stripes of black melanophores and yellow xanthophores are a prominent feature of the adult pigment pattern. In fms mutant zebrafish, however, xanthophores fail to develop and melanophore stripes are severely disrupted. fms encodes a type III receptor tyrosine kinase expressed by xanthophores and their precursors and is the closest known homologue of kit, which has long been studied for roles in pigment pattern development in amniotes. In this study we assess the cellular and temporal requirements for Fms activity in promoting adult pigment pattern development. By transplanting cells between fms mutants and either wild-type or nacre mutant zebrafish, we show that fms acts autonomously to the xanthophore lineage in promoting the striped arrangement of adult melanophores. To identify critical periods for fms activity, we isolated temperature sensitive alleles of fms and performed reciprocal temperature shift experiments at a range of stages from embryo to adult. These analyses demonstrate that Fms is essential for maintaining cells of the xanthophore lineage as well as maintaining the organization of melanophore stripes throughout development. Finally, we show that restoring Fms activity even at late larval stages allows essentially complete recovery of xanthophores and the development of a normal melanophore stripe pattern. Our findings suggest that fms is not required for establishing a population of precursor cells during embryogenesis but is required for recruiting pigment cell precursors to xanthophore fates, with concomitant effects on melanophore organization.  (+info)

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