Cytophotometric determination of the binding of basic dyes by DNA following acetylation

Summary DNA was removed from various tissues by histochemical acetylation of amino groups in proteins using pure acetic anhydride, as demonstrated by cytophotometric (UV, Feulgen, gallocyanin chromalum) and biochemical techniques. Since new phosphate groups were simultaneously exposed, the intensity of methylene blue staining was increased in spite of the nucleic acid release. Under conditions where no extraction occurs the staining intensity increases for more than 30 per cent. On the other hand, the staining intensity of gallocyanin chromalum kept constant. As it had been demonstrated previously, that gallocyanin chromalum binds to about 86 per cent of the DNA phosphate groups, it was concluded that this dye binds to a higher percentage of phosphate groups than do the usual basic dyes. Since it is not possible under the conditions used to make all nucleic acid phosphate groups available for basic dye binding by blocking the amino groups of proteins it can be assumed that not only electrostatic, but also spatial and steric relationships influence the binding capacity of basic dyes to the phosphate groups of nucleoproteins.Supported by a grant from the Deutsche Forschungsgemeinschaft, Bad Godesberg, Germany.     

Binding of methylene blue to alginate: Effect of acid treatment on metachromasy

The spectral properties of methylene blue (MB) in solutions of Na alginate depend on the severity of prior acid treatment of the polysaccharide. The spectral properties affected are the fraction of MB in monomeric form, the relative amounts of metachromatic dye absorbing near 570 and near 595 nm, and the intensity and sign of circular dichroism (CD) activity associated with the 570 nm bands, at various ratios of polymer equivalents to dye (P/D) from 1300 to 4. Acid treatment consisted of reaction of dry, alcohol-precipitated and presumably native alginate with 0·3m HCl at room temperature for 5 min to 8 h. Acid-induced changes showed immediate (5 min) and slow (4–8 h) stages. In both stages the fractions of MB in monomeric form and in the 595 nm metachromatic form increased. CD activity was little affected by brief acid treatment (except in range P/D=165 to 55), but diminished at all P/D values on prolonged acid treatment. Minor changes were observed in the infrared spectra of alginate films. Fresh alcohol-precipitated alginate, untreated with acid, did not precipitate when dye was in excess, nor did it form gel beads in CaCl₂ solution. It is concluded that dilute acid treatment alters the stereospecific properties of native alginate, perhaps by inducing conformational changes in the constituent copolymer segments.     

Effects of Yariv dyes, arabinogalactan-protein binding reagents, on the growth and viability of Brazilian pine suspension culture cells

Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins implicated in several aspects of plant growth and development. (β-d-glucosyl)₃ Yariv phenylglycoside (β-GlcY), commonly known as Yariv reagent, selectively binds AGPs. We treated cell suspension cultures of Araucaria angustifolia, the Brazilian pine, with β-GlcY and observed inhibition of biomass increase in a culture medium with 50 μM β-GlcY. However, the growth was not inhibited by (α-d-galactosyl)₃ Yariv phenylglycoside (α-GalY) which does not bind AGPs. Fluorescein diacetate staining of cells indicated that β-GlcY severely affected cell viability. However, cell swelling, bursting and release of cellular contents, all characteristics of necrotic cell death, were not observed in β-GlcY-treated cells. Instead, programmed cell death (PCD) structural changes such as cytoplasmic shrinkage and condensation were observed in β-GlcY-treated cells. In addition, callose accumulation, which is another marker of PCD, was also observed in β-GlcY-treated cells. The use of both, Ac-VEID-CHO, an inhibitor of caspase-like proteolytic activity related to PCD, and phenyl methyl sulphonyl fluoride (PMSF), a protease inhibitor known to suppress PCD, in the culture medium did not reverse the growth inhibition caused by β-GlcY. These data indicate that the β-GlcY-induced inhibition of Araucaria cell’s growth is related to AGP perturbation, and also that this growth inhibition is due to increased cell death not driven by necrosis.     

Photo-sensitivity of oxidised cholesterol black lipid membrane incorporated with acid and basic dyes

Summary The dark resistance and capacitance of oxidised cholesterol Black Lipid Membrane was measured in presense of acidic and basic dyes. The resistance of the membrane decreases almost 10-fold while the capacitance increases with increasing concentration of dyes. The results have been interpreted in terms of solubilization of the dyes by the Black Lipid Membrane. Exposure of the BLMs containing dyes to visible light filtered through 5 % CuSO₄ solution, produced trans membrane emf. These photo-emfs are attributed to the production of charge carriers and their subsequent separation. Similarities between the dye modified BLMs and dye coated electrodes have been noted.These investigations were supported by grants from the U.S. National Institutes of Health and the Office of Saline Water.The author is thankful to Professor Dr. H. T. Tien for his helpful suggestions during this work.     

Effects of temperature and pH on adsorption of basic brown 1 by the bacterial biopolymer poly(gamma-glutamic acid)

Poly(gamma-glutamic acid) (gamma-PGA), an extracellular polymeric substance (EPS) synthesized by Bacillus species, was explored to study its interaction with the basic brown 1 dye by conducting a systematic batch adsorption study as affected by two critical parameters, temperature and pH. Adsorption isotherms were closely predicted by Temkin equation among the eight isotherm models tested. The rate of adsorption was very rapid attaining equilibrium within 60 min and the kinetics were well described by both modified second-order and pseudo second-order models. Boyd's ion exchange model, which assumes exchanges of ions to be a chemical phenomenon, also fitted the kinetic data precisely. The adsorption rate increased with increasing solution temperature, however, a reversed trend was observed for the adsorption capacity. Changes in enthalpy, entropy and free energy values revealed dye adsorption by gamma-PGA to be an exothermic and spontaneous process involving no structural modification in gamma-PGA, whereas the activation energy of 37.21 kJ/mol indicated dye adsorption to be reaction-controlled. Following a rise in solution pH, the dye adsorption increased and reached a plateau at pH 5, while the maximum release of dye from spent gamma-PGA occurred at pH 1.5, suggesting a possible ion exchange mechanism. Ion exchange adsorption of basic dyes by gamma-PGA was further proved by the presence of two new IR bands at approximately 1600 and 1405.72 cm(-1), representing asymmetric and symmetric stretching vibration of carboxylate anion, for dye-treated gamma-PGA.     

Conformational and binding properties of chicken liver basic fatty acid binding protein in solution

The conformation of basic fatty acid binding protein from chicken liver and the binding properties of the apo protein toward 11-dansylamino-undecanoic acid were investigated by CD and fluorescence spectroscopy. In one set of experiments the binding process was followed by the appearance of induced optical activity in the absorption region of the dansyl chromophore. In a second set of experiments the binding process was followed by the large enhancement of emission fluorescence of the dansyl fluorophore. From the saturation curves, the stoichiometry of the complex and the binding constant of the fatty acid to the protein were precisely determined. The values of the dissociation constant determined with the two methods were in excellent agreement: we obtained K_D = (1.0 ± 0.1) · 10^?6M in a 0.9 : 1 stoichiometry. The native conformation of the protein is remarkably stable in a variety of solvent systems, including acetonitrile–water, ethylene glycol–water, and dicxane–water of various compositions. The CD results also showed that the binding of the fatty acid does not induce any appreciable change in the protein conformation. In a mixture of water and 2,2,2-trifluoroethanol 1 : 9 (v/v), the native conformation collapses and a new ordered structure is formed, characterized by a high amount of α-helix. © 1994 John Wiley & Sons, Inc.     

Effects of inter-annual temperature variability,including ENSO and post-ENSO events,on reproductive traits in the tunicate Pyura praeputialis

Reproduction in tunicates is considered to be particularly vulnerable to changes in seawater temperature. In the present study we investigated the effects of sea surface temperature and temperature anomalies on reproductive traits of the non-native sessile tunicate Pyura praeputialis. Reproductive traits of this species were investigated over the course of 67 months, based on samples collected at two localities (eastern and western shorelines) of the Bay of Antofagasta. The study period included years with different oceanic and atmospheric conditions: a warm event, El Niño (June 1997 to July 1998); a cold event, La Niña (August 1998 to December 2000); and post-La Niña (January 2000 to December 2002). We compared two common indices (condition and gonadosomatic) and histological sections to evaluate the maximum reproduction index of this introduced species that dominates a large part of the rocky intertidal habitat in the Bay of Antofagasta. We found sexually mature individuals all year-round and a decrease in both reproductive indices matching the reduction in the sea surface temperature during the austral autumn and winter months. The results suggest that gonad development in this species is sensitive to sea surface temperature and thermal anomalies. We conclude that future thermal anomalies or projected global average sea surface warming associated with climate change might have no negative consequences on these reproductive traits of P. praeputialis. This suggests this species is ecologically resilient and that the ecosystem services that this species provides for other invertebrate and algal species will be maintained.