Interactions involving the cyanine dye,diS-C3-(5), cytochromec and liposomes and their implications for estimations of Δψ in cytochromec oxidase-reconstituted proteoliposomes

Summary The interference of cytochromec with absorption and fluorescence changes of the cyanine dye, diS-C₃-(5), was investigated in the presence of liposomes and cytochromec-oxidase reconstituted proteoliposomes. The apparent cytochromec-dependent quenching of diS-C₃-(5) fluorescence, and the associated absorbance losses in the presence of liposomes and proteoliposomes in low ionic strength media, are due to destruction of the dye caused by cytochromec-mediated lipid peroxidation. The rate of this reaction was further enhanced in the presence of hydrogen peroxide. Even in the absence of liposomes or proteoliposomes, a cytochromec-induced breakdown of dye was observed in the presence of hydrogen peroxide.The cytochromec mediated absorbance and fluorescence losses of diS-C₃-(5) in liposomal or proteoliposomal systems are prevented by Ca²⁺ and La³⁺ ions, by ascorbate, by high ionic strength and by the antioxidant BHT. Under these conditions, the process of lipid peroxidation mediated by cytochromec is inhibited either directly (e. g. by BHT) or indirectly, by preventing the binding of cytochromec to lipid vesicles. The impact of these findings upon the experimental estimation of membrane potential inaa ₃-reconstituted proteoliposomes is discussed.     

Impermeant potential-sensitive oxonol dyes: III. The dependence of the absorption signal on membrane potential

Summary We have measured potential-dependent changes in the absorption of light by oxidized cholesterol bilayer lipid membranes in the presence of impermeant oxonol dyes. The magnitude of the absorption signal increased linearly with the size of potential steps over a range of 500 mV. The signal also increased when the offset voltage of the pulse train was increased from –150 to +150 mV. The data are consistent with the on-off mechanism proposed by E. B. George et al. (J. Membrane Biol.103:245–253, 1988) in which the probe undergoes potential-dependent movement between a binding site in the membrane and an aqueous region just off the surface of the membrane. An equilibrium thermodynamic analysis of the experimental data indicates that the negatively charged oxonol chromophore senses only 5–10% of the total membrane potential difference across the membrane when it is driven into a nonpolar binding site on the membrane.ASW, Carnegie-Mellon University     

Impermeant potential-sensitive oxonol dyes: II. The dependence of the absorption signal on the length of alkyl substituents attached to the dye

Summary We have measured the potential-dependent light absorption changes of 43 impermeant oxonol dyes with an oxidized cholesterol bilayer lipid membrane system. The size of the signal is strongly dependent on the chain length of alkyl groups attached to the chromophore. Dye molecules with intermediate chain lengths give the largest signals. To better understand the dependence of the absorbance signal on alkyl chain length, a simple equilibrium thermodynamic analysis has been derived. The analysis uses the free energy of dye binding to the membrane and the on-off model (E.B. George et al.,J. Membrane Biol.,103:245–253, 1988a) for the potential-sensing mechanism. In this model, a population of dye molecules in nonpolar membrane binding sites is in a potential-dependent equilibrium with a second population of dye that resides in an unstirred layer adjacent to the membrane. Dye in the unstirred layer is in a separate equilibrium with dye in the bulk bathing solution. The equilibrium binding theory predicts a sigmoidally shaped increase in signal with increasing alkyl chain length, even for very nonpolar dyes. We suggest that aggregation of the more hydrophobic dyes in the membrane bathing solution may be responsible for their low signals, which are not predicted by the theory.     

Binding of textile azo dyes byMyrothecium verrucaria

Summary A strain ofMyrothecium verrucaria that showed a high capacity for rapid decolorization of textile dye solutions was isolated from soil. As much as 70%, 86%, and 95% of Orange II, 10B (blue) and RS (red) dyes (color index no. 15510, 20470, 23635), respectively, were adsorbed from solutions of approximately 0.2 g dye per liter in 5 h by approximately 4.5 g dry weight of cells per liter of dye solution. Intact cells showed a higher adsorption capacity than disrupted cells for Orange II and RS but not for 10B. Dye bound to cells was recoverable by extraction with methanol and methanol-treated cells were able to be recycled, albeit with a slightly diminished dye-binding capacity. The Tween detergents were shown to reduce dye adsorption. Dyes strongly bound to the fungal biomass required sonication in dH₂O or in Triton X-100 or extraction with methanol for their removal. These results suggest that hydrophobic/hydrophilic interactions are important in dye binding.     

Enhanced decolorization of sulfonated azo dye methyl orange by single and mixed bacterial strains AK1, AK2 and VKY1

Abstract

Methyl orange, a sulfonated azo dye having various industrial applications was decolorized by three bacteria Bacillus sp. strain AK1, Lysinibacillus sp. strain AK2 and Kerstersia sp. strain VKY1. The effect of various factors such as dye concentration, pH, temperature and NaCl concentration on decolorization was investigated. At 200?mg/L methyl orange concentration, the strains AK1, AK2 and VKY1 exhibited maximum decolorizing potential of 93, 95 and 96%, respectively, at temperature 35?°C and pH 7.0 within 18?h of incubation. These strains decolorized the dye over a wide range of pH (5–10), temperature (15–55?°C), and NaCl concentration (5–20?g/L). Further, these strains decolorize up to 800?mg/L concentrations of methyl orange within 24?h. The dye decolorization efficiency was further increased by using different consortia of these three strains which could decolorize the dye completely within 12?h of incubation. The cell-free extracts of the strains AK1, AK2 and VKY1 grown on methyl orange exhibited the azoreductase activity of 0.4794, 1.56 and 1.01?µM/min/mg protein, respectively. HPLC and FTIR analysis of the dye decolorized sample indicated the formation of 4-aminobenzenesulfonic acid and N,N-dimethyl-p-phenylenediamine as breakdown products of azo bond. The high decolorization potential of these bacterial strains individually and in consortia has potential application in remediation of dye effluent.     

Bacterial community dynamics involved in Reactive Orange M2R dye degradation using a real time quantitative PCR and scale up studies using sequence batch reactor

Bacterial consortium volatile suspended soil (VSS) (Vatva soil sample) with a capability of azo dye Reactive Orange M2R (ROM2R) decolorization and degradation (shown in our earlier studies using Fourier transform infrared spectroscopy (FTIR) and phytotoxicity studies) was isolated from industrial wastewaters by enrichment culture technique. The present study was carried out to study bacterial population dynamics in consortium Vatva soil sample (VSS) during azo dye ROM2R degradation and to identify the consortium members that were actively involved in the degradation process. To achieve this goal, a real-time Polymerase chain reaction (PCR) assay targeting species-specific region of 16S rDNA of each consortial bacteria was developed to provide quantitative information about the bacterial abundance during azo dye degradation. The real-time PCR assay indicated that Pseudomonas aeruginosa (VSS-6) dominated consortium bacterial community during the active continuous bioremediation process. Attempt has been made to scale up from 100 ml volume to 10 L operation volume with intermittent additions (batch fed loadings) in a Sequence batch reactor (SBR). The development of VSS consortium biomass (MLVSS), changes in COD and biochemical oxygen demand, and the dye degradation were studied under conditions simulating the operations of biological effluent treatment in an attempt to develop a commercially applicable dye effluent treatment process unit.     

A mitochondria-selective near-infrared-emitting fluorescent dye for cellular imaging studies

This communication details the synthesis, evaluation of photophysical properties, and cellular imaging studies of cyanine chromophore based fluorescent dye 1 as a selective imaging agent for mitochondria.     

Localization of calcium during somatic embryogenesis of carrot (Daucus carota L.)

Summary The distribution of free cytosolic Ca²⁺ was studied during somatic embryogenesis of carrot using confocal scanning laser microscopy with fluo-3 as a fluorescent Ca²⁺ indicator. Chlorotetracycline fluorescence, antimonate precipitation and proton induced X-ray emission analysis were used as additional methods to confirm the results obtained with fluo-3. The process of embryogenesis was found to coincide with a rise in the level of free cytosolic Ca²⁺. The level of Ca²⁺ was low in proembryogenic masses and relatively high in later stages of embryogenesis. The highest signal was found in the protoderm of embryos from the late globular to the torpedo-shaped stage. A gradient in fluorescence intensity was often observed along the longitudinal axis of the embryos. The most conspicuous intracellular signal was found in the nucleus. Other organelles did not take up the dye and were always without fluorescence. The changes in [Ca²⁺]_c are discussed in relation to physiological processes which are known to be important during somatic embryogenesis.This article is dedicated to the memory of the late Dr. Hans-Dieter Reiss.     

Regulation of cardiac gap junction channel permeability and conductance by several phosphorylating conditions

Short term (15 min) effects of activators of protein kinase A (PKA), PKC and PKG on cardiac macroscopic (g_j) and single channel (_j) gap junctional conductances were studied in pairs of neonatal rat cardiomyocytes. Under dual whole-cell voltage-clamp, PKC activation by 100 nM TPA increased g_j by 16 ± 2% (mean ± S.E.M, n=9), 1.5 mM of the PKG activator 8-bromo-cGMP (8Br-cGMP) decreased g_j by 26 ± 2% (n=4), whereas 1.5 mM of the PKA activator 8Br-cAMP did not affect g_j (1 ± 5%, n=11). Single cardiac gap junction channel events, resolved in the presence of heptanol, indicated two _j sizes of 20 pS and 40–45 pS. Under control conditions, the larger events were most frequently observed. Whereas 8Br-cAMP did not change this distribution, TPA or 8Br-cGMP shifted the _j distribution to the lower sizes. Diffusion of 6-carboxyfluorescein (6-CF), a gap junction permeant tracer, from the injected cell to neighboring cells was studied on small clusters of neonatal rat cardiomyocytes. Under control conditions, 6-CF labeled 8.4 ± 0.4 cells (mean ± S.E.M, n=31). Whereas 8Br-cAMP did not change the extent of dye transfer (8.1 ± 0.5 cells, n=10), TPA restricted the diffusion of 6-CF to 2.2 ± 0.2 cells (n=30) and 8Br-cGMP to 3.5 ± 0.3 cells (n=10). This suggests that permeability and single channel conductance of Cx43 gap junction channels are parallel related. Altogether, these results point to the differential modulation of electrical and metabolic coupling of cardiac cells by various phosphorylating conditions.