Study of the integument that covering back and stinger of the freshwater stingray Potamotrygon rex (Chondricthyes, Potamotrygonidae).

The objective of this study was to describe the histology and histochemistry of the integument covering the back and stinger of the freshwater stingray Potamotrygon rex, endemic to the Middle Upper Tocantins River. The species has a dark back and yellowish circular spots that extend to the tail, which has one to two stings located in the median portion of the tail. Through histological study it was observed that the epithelia of the back and stinger are composed of non-keratinized stratified pavement epithelial tissue, and are organized in three layers: basal, intermediate and superficial. The differences between the tissues are related to the cell types that compose them. The back is organized with epithelial cells, mucus cells, granulocyte cells and chromatophores. The mucus cells are distributed in different layers along the animal's back, influencing the thickness of the tissue. The tissue that covers the stinger is composed of epithelial cells, chromatophores and specialized cells in protein synthesis. In the histochemistry, the stinger epithelial cells were stained with Bromophenol Blue, especially those of the intermediate layer, which were called specialized cells. In the back the epithelial cells were stained with Bromophenol Blue, Alcian Blue and PAS, and the mucous cells with PAS. In both tissues the presence of protein reserves was detected, areas so called because they are stained strongly with Bromophenol Blue. The results show that the stinger presents activity directed to the production of proteins, and that the back is organized to produce different components, which constitute the cuticle that covers the animal's body.

Characterization of a thermotolerant and acidophilic mannanase producing Microbacterium sp. CIAB417 for mannooligosachharide production from agro-residues and dye decolorization.

Mannanases are ubiquitous enzymes and are being explored for diverse industrial applications. In this study, a novel bacterial strain Microbacterium sp. CIAB417 was identified and characterized for extracellular production of mannanase. Microbacterium sp. CIAB417 was found to produce maximum mannanase after 36 h of incubation at 37 °C. Mannanase produced by the isolate was observed for maximum activity at optimum pH of 6 and optimum temperature of 50 °C. Crude mannanase was found to be capable of producing mannooligosachharides (MOS) by hydrolyzing hemicellulose from locust bean gum and Aloe vera. The produced MOS was characterized and found to be mixture of mannobiose to mannohexose units. Mannanase was also explored for decolorization of dyes. Bromophenol blue and coomassie blue R-250 were observed to be decolorized to the extent of 45.40 and 42.75%, respectively. Hence, the identified bacterial strain producing mannanase could be of great significance for applications in food and textile industry.

Double cuticle barrier in two global pests, the whitefly Trialeurodes vaporariorum and the bedbug Cimex lectularius.

The integument protects the organism against penetration of xenobiotics and water that would potentially interfere with homeostasis. In insects that play key roles in a variety of agricultural and ecological habitats, this inward barrier has barely been investigated. In order to advance knowledge in this field, we studied integumental barrier (cuticle) permeability in the two global pests Trialeurodes vaporariorum (greenhouse whitefly) and Cimex lectularius (bedbug), applying a simple dye-penetration assay. In agreement with our recent findings in Drosophila melanogaster, we show that the surface of these insects is regionalised. We also show that, in contrast to the single barrier in D. melanogaster, two barriers with distinct temperature-sensitive and lipid-based physico-chemical material properties act in parallel to protect these insects against penetration of hydrophilic molecules. These findings imply the existence of unexplored mechanisms by which the cuticle acts as a protective coat against the penetration of water and xenobiotics, including pollutants and insecticides.

Purification and characterization of extracellular laccase produced by Ceriporiopsis subvermispora and decolorization of triphenylmethane dyes.

Laccases of white-rot fungi provide a promising future as a tool to be used in the field of biodegradation of synthetic dyes with different chemical structures. The aim of this study was production, characterization, and application of laccases from the white-rot fungus Ceriporiopsis subvermispora ATCC 90467 for decolorization of triphenylmethane dyes that could remain persistent in wastewater. Laccase was purified from a C. subvermispora culture by a four-step method resulting high specific activity of 2,571 U g-1 , 88-fold higher than crude laccase. Purified laccase (molecular weight 45 kDa) had the optimum activity at pH 2.0 and the optimum temperature 50 °C using ABTS as chromogenic substrate. Laccases efficiently decolorized triphenylmethane dyes such as Malachite Green (87.8%), Bromocresol Purple (71.6%), and Methyl Violet (68.1%) without redox mediator. However, decolorization percentage of hardly degradable triphenylmethane dyes such as Phenol Red, Bromophenol Blue, and Brilliant Blue R-250 was increased the presence of some low-molecular weight compounds (natural or synthetic redox mediators). Purified laccases were resistant to Mg2+ , Ca2+ , Ba2+ , Mn2+ , Fe2+ , Cu2+ , Zn2+ , and Sn2+ (10 mmol L-1 ). These findings suggest that laccases from C. subvermispora are able to decolorize triphenylmethane dyes without the negative influence of metal ions that can be found in wastewater.

Development of an ion-pair to improve the colon permeability of a low permeability drug: Atenolol.

To ensure the optimal performance of oral controlled release formulations, drug colon permeability is one of the critical parameters. Consequently developing this kind of formulations for low permeability molecules requires strategies to increase their ability to cross the colonic membrane. The objective of this work is to show if an ion-pair formation can improve the colon permeability of atenolol as a low permeability drug model. Two counter ions have been tested: brilliant blue and bromophenol blue. The Distribution coefficients at pH7.00 (DpH7) of atenolol, atenolol + brilliant blue and atenolol + bromophenol blue were experimentally determined in n-octanol. Moreover, the colonic permeability was determined in rat colon using in situ closed loop perfusion method based in Doluisio's Technique. To check the potential effects of the counter ions on the membrane integrity, a histological assessment of colonic tissue was done. The results of the partitioning studies were inconclusive about ion-pair formation; nevertheless colon permeability was significantly increased by both counter ions (from 0.232±0.021cm/s to 0.508±0.038cm/s in the presence of brilliant blue and to 0.405±0.044cm/s in the presence of bromophenol blue). Neither damage on the membrane was observed on the histological studies, nor any change on paracellular permeability suggesting that the permeability enhancement could be attributed to the ion-pair formation.

A subset of neurons controls the permeability of the peritrophic matrix and midgut structure in Drosophila adults.

The metazoan gut performs multiple physiological functions, including digestion and absorption of nutrients, and also serves as a physical and chemical barrier against ingested pathogens and abrasive particles. Maintenance of these functions and structures is partly controlled by the nervous system, yet the precise roles and mechanisms of the neural control of gut integrity remain to be clarified in Drosophila Here, we screened for GAL4 enhancer-trap strains and labeled a specific subsets of neurons, using Kir2.1 to inhibit their activity. We identified an NP3253 line that is susceptible to oral infection by Gram-negative bacteria. The subset of neurons driven by the NP3253 line includes some of the enteric neurons innervating the anterior midgut, and these flies have a disorganized proventricular structure with high permeability of the peritrophic matrix and epithelial barrier. The findings of the present study indicate that neural control is crucial for maintaining the barrier function of the gut, and provide a route for genetic dissection of the complex brain-gut axis in adults of the model organism Drosophila.

Reductive Dehalogenation of Oligocyclic Phenolic Bromoaromatics by Dehalococcoides mccartyi Strain CBDB1.

Dehalococcoides mccartyi strains transform many halogenated compounds and are used for bioremediation. Such anaerobic transformations were intensively studied with chlorinated and simply structured compounds such as chlorinated benzenes, ethenes, and ethanes. However, many halogenated oligocyclic aromatic compounds occur in nature as either naturally produced materials or as part of commercial products such as pharmaceuticals, pesticides, or flame retardants. Here, we demonstrate that the D. mccartyi strain CBDB1 reductively debrominated two oligocyclic aromatic phenolic compounds, tetrabromobisphenol A (TBBPA) and bromophenol blue (BPB). The strain CBDB1 completely converted TBBPA to bisphenol A and BPB to phenol red with a stepwise removal of all bromide substituents. Debromination (but no cell growth) was detected in the cultures cultivated with TBBPA. In contrast, strain CBDB1 grew when interacting with BPB, demonstrating that this substrate was used as an electron acceptor for organobromine respiration. High doses of BPB delayed debromination and inhibited growth in the early cultivation phase. A higher toxicity of TBBPA compared with that of BPB might be due to the higher lipophilicity of TBBPA. Mass spectrometric analyses of whole-cell extracts demonstrated that two proteins encoded by the reductive dehalogenase homologous genes CbdbA1092 and CbdbA1503 were specifically induced by the used oligocyclic compounds, whereas others (e.g., CbdbA84 (CbrA)) were downregulated.

Triphenylmethane dyes, an alternative for mediated electronic transfer systems in glucose oxidase biofuel cells.

The bioelectrochemical behavior of three triphenylmethane (TPM) dyes commonly used as pH indicators, and their application in mediated electron transfer systems for glucose oxidase bioanodes in biofuel cells was investigated. Bromophenol Blue, Bromothymol Blue, Bromocresol Green were compared bioelectrochemically against two widely used mediators, benzoquinone and ferrocene carboxy aldehyde. Biochemical studies were performed in terms of enzymatic oxidation, enzyme affinity, catalytic efficiency and co-factor regeneration. The different features of the TPM dyes as mediators are determined by the characteristics in the oxidation/reduction processes studied electrochemically. The reversibility of the oxidation/reduction processes was also established through the dependence of the voltammetric peaks with the sweep rates. All three dyes showed good performances compared to the FA and BQ when evaluated in a half enzymatic fuel cell. Potentiodynamic and power response experiments showed maxima power densities of 32.8 µW cm(-2) for ferrocene carboxy aldehyde followed by similar values obtained for TPM dyes around 30 µW cm(-2) using glucose and mediator concentrations of 10 mmol L(-1) and 1.0 mmol L(-1), respectively. Since no mediator consumption was observed during the bioelectrochemical process, and also good redox re-cycled processes were achieved, the use of triphenylmethane dyes is considered to be promising compared to other mediated systems used with glucose oxidase bioanodes and/or biofuel cells.

Bioprocess of triphenylmethane dyes decolorization by Pleurotus ostreatus BP under solid-state cultivation.

With an aim to evaluate dye decolorization by white rot fungus on natural living conditions, reproducing by solidstate fermentation, the process of triphenylmethane dyes decolorization using the white rot fungus P. ostreatus BP, cultivated on rice straw solid-state medium, has been demonstrated. Three typical dyes, including malachite green, bromophenol blue, and crystal violet, were almost completely decolorized by the fungus after 9 days of incubation. During the process of dye decolorization, the activities of enzyme secreted by the fungus, and the contents of soluble components, such as phenolic compounds, protein, and sugar, changed regularly. The fungus could produce ligninolytic, cellulolytic, and hemicellulolytic enzymes and laccase was the most dominant enzyme in solid-state medium. Laccase, laccase isoenzyme, and the laccase mediator could explain the decolorization of malachite green, bromophenol blue, and crystal violet by the fungus in solid medium, respectively. It is worth noting that the presence of the water-soluble phenolic compounds could stimulate the growth of fungus, enhance the production of laccase, and accelerate dye decolorization.

Study on the interaction mechanism of lysozyme and bromophenol blue by fluorescence spectroscopy.

The interaction of lysozyme with bromophenol blue (BPB) in acetate buffer (pH 6.0) was studied by fluorescence quenching method for the first time. It was found that BPB could conspicuously quench the fluorescence of lysozyme by the static quenching process, possibly due to the binding on the active site near Trp62. The binding parameters including the binding constant and the number of binding site were calculated. The thermodynamic parameters DeltaH degrees, DeltaS degrees and DeltaG degrees at different temperatures were obtained. The formation of lysozyme-BPB complex depended on the cooperation of the hydrophobic and electrostatic forces. And the binding average distance between lysozyme and BPB was determined. The effect of common metal ions on the binding constant of lysozyme-BPB was also examined.

Delivery of several forms of DNA, DNA-RNA hybrids, and dyes across human sclera by electrical fields.

PURPOSE: Iontophoresis has been used for drug delivery across the cornea for many years. We sought to test whether small charged dyes and DNA can be transferred across human sclera by an electric field. METHODS: Full-thickness human scleral fragments were embedded vertically in an agarose gel and positioned to completely span individual gel lanes. The scleral fragments were located approximately 1 cm downstream from the gel wells. DNA or dyes were loaded into the wells and electrophoresis was carried out at about 3.3 V/cm for approximately 2 h per run. Movement of DNA and dyes through the agarose and sclera was measured with either digital time-lapse photography or through DNA extraction and purification from the gel. SYBR green stain was used as a sensitive method to detect DNA. RESULTS: Digital time-lapse photography of agarose gel electrophoresis revealed that two dyes, xylene cyanol and bromphenol blue, passed through the sclera in the presence of an electric field. Xylene cyanol was driven through the sclera virtually unimpeded except for some spreading of the dye. Bromphenol blue was slowed markedly by the sclera, but it too eventually passed through the tissue. Small DNAs, including a single stranded 51-mer and a double hairpin 68-mer oligonucleotide, passed through the sclera as detected by SYBR green staining. Linear double stranded DNAs ranging from 50 bp to 12,000 bp passed through the sclera. The larger the DNA, the slower the rate of passage through the sclera, and the greater the band spreading. pEGFP-1 (a 3 kb plasmid) passed through the sclera but was accompanied by a great amount of band spreading. Following completion of the initial electrophoresis run, the plasmid DNA was extracted from the smeared bands in the agarose distal to the sclera and re-run on a second gel without sclera. The initially smeared plasmid bands resolved into 2 distinct bands after extraction and purification and matched well with control plasmid bands. CONCLUSIONS: Charged molecules such as xylene cyanol, bromphenol blue, and DNAs ranging from 51 bp oligonucleotides to a 3 kb plasmid can be driven across human sclera by an electric field and directly detected. Passage of plasmids was efficient, but the plasmid bands were diffuse after transit. This technique offers promise as a noninvasive DNA delivery tool, where gene therapy can be accomplished by small RNA or DNA synthetic oligonucleotides, larger double stranded fragments, or even plasmids.

Purification and characterization of an aflatoxin degradation enzyme from Pleurotus ostreatus.

Nineteen fungi were tested for their ability to degrade aflatoxin B1 (AFB1). An extracellular enzyme from the edible mushroom Pleurotus ostreatus showed afaltoxin-degradation activity detected by thin-layer chromatography (TLC). An enzyme with this activity was purified by two chromatographies on DEAE-Sepharose and Phenyl-Sepharose. The apparent molecular mass of the purified enzyme was estimated to be 90 kDa by SDS-PAGE. Optimum activities were found in the pH range between 4.0 and 5.0 and at 25 degrees C. Also, degradation activity of several dyes in the presence of H2O2 was tested, resulting in the detection of bromophenol blue-decolorizing activity. Based on these data, we suggest this enzyme is a novel enzyme with aflatoxin-degradation activity. Fluorescence measurements suggest that the enzyme cleaves the lactone ring of aflatoxin.

Selective staining of proteins with hydrophobic surface sites on a native electrophoretic gel.

Chemical proteomics aims to characterize all of the proteins in the proteome with respect to their function, which is associated with their interaction with other molecules. We propose the identification of a subproteomic library of expressed proteins whose native structures are typified by the presence of hydrophobic surface sites, which are often involved in interactions with small molecules, membrane lipids, and other proteins, pertaining to their functions. We demonstrate that soluble globular proteins with hydrophobic surface sites can be detected selectively by staining on an electrophoretic gel run under nondenaturing conditions. The application of these staining techniques may help elucidate new catalytic, transport, and regulatory functionalities in complex proteomic screenings.

The identification of hydrophobic sites on the surface of proteins using absorption difference spectroscopy of bromophenol blue.

Hydrophobic sites on the surface of protein molecules are thought to have important functional roles. The identification of such sites can provide information about the function and mode of interaction with other cellular components. While the fluorescence enhancement of polarity-sensitive dyes has been useful in identifying hydrophobic sites on a number of targets, strong intrinsic quenching of Nile red and ANSA dye fluorescence is observed on binding to a cytochrome c('). Fluorescence quenching is also observed to take place in the presence of a variety of other biologically important molecules which can compromise the quantitative determination of binding constants. Absorption difference spectroscopy is shown not to be sensitive to the presence of fluorescence quenchers but sensitive enough to measure binding constants. The dye BPB is shown to bind to the same hydrophobic sites on proteins as polarity-sensitive fluorescence probes. The absorption spectrum of BPB is also observed to be polarity sensitive. A binding constant of 3x10(6)M(-1) for BPB to BSA has been measured by absorption difference spectroscopy. An empirical correlation is observed between the shape of the absorption difference spectrum of BPB and the polarity of the environment. The results indicate that absorption difference spectroscopy of BPB provides a valuable supplement to fluorescence for determining the presence of hydrophobic sites on the surface of proteins as well as a method for measuring binding constants.

On the role of lysine residues in the bromophenol blue-albumin interaction.

In order to explore the role of buried lysine residues of bovine serum albumin (BSA) in its interaction with bromophenol blue (BPB), three acetylated derivatives of albumin namely: 90%, 100% and 10%/chiefly having modification of buried lysine residues) were prepared by conventional and double modification techniques. The modification of lysine residues resulted in the change in conformation, as evidenced by the increase in Stokes radius from 3.55 nm (for native albumin) to 4.91 and 4.97 nm for 90% and 100% acetylated albumins, respectively. Modification of buried lysine residues (10% acetylated preparation) of albumin increased the Stokes radius up to 3.96 nm. The interaction of BPB with albumin preparations was studied spectrophotometrically at ionic strength 0.4 and at three different pH values i.e., 4.0, 6.0 and 8.0. There was decrease in BPB binding on increasing the modification. A decrease of 63% and 69% was noticed at pH 8.0 in 90% and 100% acetylated preparation, respectively. The 10% acetylated BSA preparation with minimum conformational changes also showed a significant decrease (31%) in BPB binding at pH 8.0. The change in Kd from 2.04 x 10(-6) M for native albumin to 5.41 x 10(-6) M for 100% acetylated albumin and 3.39 x 10(-6) M for 10% acetylated preparation at pH 8.0 confirmed the critical role of buried lysine residues in BPB-BSA interaction.

Interaction between Asp-85 and the proton-releasing group in bacteriorhodopsin. A study of an O-like photocycle intermediate.

Upon light adaptation by continuous (or pulsed) illumination, the artificial bacteriorhodopsin (bR) pigments, I and II, derived from synthetic 14F retinal and a short polyenal, respectively produce a long-lived red-shifted species denoted O1. An analogous phenomenon was observed by Sonar, S., et al. [(1993) Biochemistry 32, 2263-2271], in the case of the Y185F mutant (pigment III). The nature of these O1 species was investigated by studying a series of effects, primarily their red light photoreversibility, the associated proton uptake and release processes, and the effects of pH on their relative amounts, which are interpreted in terms of pH-dependent acid-base equilibria. Experiments were also carried out with pigments I and II derived from the mutants D96A, E204Q, R82Q, and D85N. The O1 species of pigments I and II (and possibly also that of pigment III) are identified as an unusually long-lived (all-trans) intermediate of the photocycle of their 13-cis isomer. It is concluded that in O1, Asp-85 is protonated, a process associated with proton uptake from the extracellular side. Subsequent proton release (to the same side of the membrane) occurs from Glu-204 (or from a group closely interacting with it) prior to the decay of O1. At high pH (>9), O1 reversibly converts to a purple form, due to deprotonation of Asp-85, while at still higher pH (> 11), a blue-shifted species characterized by a deprotonated Schiff base is generated. These transitions constitute the first demonstration of the titration of a photocycle intermediate of a retinal protein. The respective pKa values are determined and discussed in relation to those pertaining to the unphotolyzed (dark-adapted) pigments. It appears that the pKa values are controlled by a hydrogen bond network involving water molecules, which binds the protonated Schiff base with Asp-85 and Glu-204. The disruption of this network in pigments I-III may also be responsible for the long lifetime of the O1 species, due to the inhibition of thermal trans-13-cis isomerization. The results are relevant to the molecular mechanism of the photocycles of both 13-cis- and all-trans-bR, primarily to the nature and to the deprotonation mechanism of the proton-releasing group.

Vanadium catalysis in bromoperoxidation reaction.

Peroxidative bromination of phenol red to its tetrabromo derivative, bromophenol blue, required vanadate in addition to H202 when carried out in the pH range of 5-7. Excess H202, with ratio of H202:vanadate of 2:1 and above, prevented the reaction. Diperoxovanadate, known to be formed in such reaction mixtures, was ineffective by itself and needed uncomplexed vanadate (V(v)) or vanadyl (V(iv)) to support bromination. Bromide-assisted reduction of the excess vanadate to vanadyl appeared to be an essential secondary reaction. In the absence of phenol red oxygen was released, and concomitantly bromide was oxidized to a form competent to brominate phenol red added after termination of oxygen release. These findings indicated participation of reactions leading to an intermediate derived from vanadyl and diperoxovanadate, previously described from this laboratory (Arch. Biochem. Biophys. 316, 319-326, 1995). Continuous bromination of phenol red occurred when glucose oxidase-glucose system was used as a source of continuous flow of H202. A scheme of reactions involving peroxovanadates (mono-, di-, mu-, and bromo-) is proposed for the formation and utilization of an active brominating species and for the recycling of the product, mono-peroxovanadate, by H202, which explains the catalytic role of vanadium in the bromoperoxidation reaction.

[Interaction of bromophenol blue with serum albumin: criteria for using dyes for assessing the state and quantity of protein]. / Vzaimodeistvie bromfenolovogo sinego s syvorotochnym al'buminom: kriterii ispol'zovaniia krasiteliia dliia otsenki sostoianiia i kolichestva belka.

The optical properties of the complexes of the pH-dependent dye bromophenol blue (BPB) with human serum albumin were investigated by the spectrophotometric method. The solvatochromic longwave displacement of bound BPB-2 absorption and BPB-1/BPB-2 redistribution were shown to form the optical signal of complexes. Because of the distortion of the bound BPB-2 signal its quantity was determined as delta A630 = A630 - A660 and the use of lambda max as structural parameter was limited to low pH less than or equal to 3. The conclusion was made that BPB is inapplicable as a structural probe on account of low structural dependence of delta A630 and pH-limitation of lambda max used. The maximal absorption delta Amax = Amax - A660 and its structural independence were obtained in the region of 70-100% occupation of the dye-binding centers of the protein. It is the optimal conditions for the quantitative determination of protein. After maximal dye binding (15-16 molecules of BPB per 1 molecule of albumin) the aggregation and precipitation of the complexes occurred.

Binding of bromophenol blue to bovine serum albumin and its succinylated forms.

Interaction of bromophenol blue with bovine serum albumin and its five succinylated forms was studied spectrophotometrically at three different ionic strengths, i.e. 0.04, 0.15 and 1.0 and at two different pH values, namely pH 7.0 and pH 5.0 respectively. Results showed a decrease in bromophenol blue binding on increasing succinylation at low ionic strengths. This decrease was more marked at pH 7.0 than pH 5.0. However, at both the pH values binding returned to a significant degree on increasing the ionic strength to 1.0. Succinylation also caused marked conformational changes at pH 7.0 and ionic strength 0.15 as evidenced by changes in hydrodynamic properties and reduction in antigen-antibody precipitin reaction. However, an increase in ionic strength to 1.0 or decrease in pH to 5.0 caused significant reversal in hydrodynamic parameters. These studies show that lysine residues of bovine serum albumin are not important in bromophenol blue binding.

[Sorption of sulfophthaleinic dyes by the brain synaptosomes of rats deprived of parodoxical sleep]. / Sorbtsiia sul'foftaleinovykh krasitelei sinaptosomami golovnogo mozga krys, lishennykh paradoksal'noi fazy sna.

The influence of paradoxical sleep deprivation on sorption of bromphenol blue, bromcresol green and bromthymol blue by rat's brain synaptosomes was studied. Effect of sleep disturbance (increase in the number of dye bindings) was shown to augment with the increase in hydrophobicity of the sulfophtaleinic dye.