Antimicrobial activities of secondary metabolites produced by endophytic fungi from Spondias mombin

We performed a search for bioactive compounds produced by fungal endophytes from Spondias mombin (Anacardiaceae). Culture broth extracts of Guignardia sp., Phomopsis sp. and Pestalotiopsis guepinii were separated by chromatographic methods and tested for biological activities. The crude extracts of these endophytes were tested against fourteen organisms, including actinomycetes, Gram-negative and Gram-positive bacteria, yeast, and filamentous fungi. All fungal extracts inhibited actinomycete growth. Guignardia sp. was also active against Escherichia coli, Staphylococcus aureus, Saccharomyces cerevisiae, Geotrichum sp., and Penicillium canadensis. Culture extracts of P. guepinii were active against S. cerevisae, while strains of Phomopsis sp. showed a pronounced antifungal effect against Cladosporium elatum, Mycotypha sp. and S. cerevisae.     

Nonlinear optical potentiometric dyes optimized for imaging with 1064-nm light

Nonlinear optical phenomena, such as two-photon fluorescence (2PF) and second harmonic generation (SHG), in combination with voltage sensitive dyes, can be used to acquire high-resolution spatio temporal maps of electrical activity in excitable cells and tissue. Developments in 1064-nm fiber laser technology have simplified the generation of high-intensity, long-wavelength, femtosecond light pulses, capable of penetrating deep into tissue. To merge these two advances requires the design and synthesis of new dyes that are optimized for longer wavelengths and that produce fast and sensitive responses to membrane potential changes. In this work, we have systematically screened a series of new dyes with varying chromophores and sidechains that anchor them in cell membranes. We discovered several dyes that could potentially be used for in vivo measurements of cellular electrical activity because of their rapid and sensitive responses to membrane potential. Some of these dyes show optimal activity for SHG; others for 2PF. This regulated approach to dye screening also allows significant insight into the molecular mechanisms behind both SHG and 2PF. In particular, the differing patterns of sensitivity and kinetics for these two nonlinear optical modalities indicate that their voltage sensitivity originates from differing mechanisms.     

Preparation and characterization of all organic NLO sol‐gel materials based on amino azobenzene dyes

A series of NLO active chromophores based on amino azobenzene dyes have been synthesized by diazotization reaction. All organic NLO‐active materials were obtained by introducing the chromophores into (hydroxymethyl)‐benzoguanamine ( HMBG ) systems by a sol‐gel process. All the sol‐gel materials exhibit excellent optical transparency. Morphology, thermal and optical properties of these polymers have been characterized. These NLO‐active polymers can be classified into reactive systems and guest‐host systems. In the reactive systems, the chromophores were condensed with HMBG in the sol‐gel process. In the guest‐host systems, the nonreactive chromophores were simply doped into HMBG matrix. A second harmonic coefficient, d₃₃, of 23.2 pm/V has been obtained. Moreover, a systematic investigation of variation of chromophoric chemical structures on NLO properties has been investigated. Covalent bonding effect between chromophore and benzoguanamine as well as steric effect are shown to greatly enhance the temporal stability for these NLO‐active sol‐gel systems.     

Chiroptical Properties of Terthiophene Chromophores Dispersed in Oriented and Unoriented Polyethylene Films

Summary: Two new chiral terthiophene chromophores II and III were prepared with 99% enantiomeric excess. Chiroptical properties of these dyes dispersed in ultra high molecular weight polyethylene (UHMWPE) films were determined and compared with the same properties in solution. In the solid state, the optical activity strongly depends on the interaction mechanisms within small crystalline aggregates of chromophores. The film morphology and chromophore dispersion were also investigated by scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). The good correlation between chromophore aggregation and chiroptical activity of the binary films promotes circular dichroism (CD) as an effective technique for monitoring the phase dispersion behaviour of dichroic dyes into polymer matrices. By mechanical stretching of the film a linearly dichroic orientation of the chromophores is obtained which results in a high degree of linear dichroism. The influence of the uniaxial orientation of terthiophene molecules along the drawing direction of UHMWPE on the chiroptical properties of the films, and the possible application of the oriented devices as linear polarizers are discussed.

Absorption and CD spectra of unoriented UHMWPE II film at different rotation angles θ.     

Monomerizing effect of caffeine, o-phenanthroline, and tannin on cationic dyes: a model system to analyze spectral characteristics of the intercalative binding to nucleic acids

If used as co-solutes in concentrated solutions of cationic planar dyes, caffeine, o-phenanthroline, and tannin induce striking hyperchromic and bathochromic shifts in their absorption spectra. Likewise, the fluorescence of acridine orange at high concentration greatly increases in the presence of caffeine, the emission peak appearing at a shorter wave-length. These spectral changes, which are similar to those produced by organic solvents, detergents, and alpha-cyclodextrin, reflect the disaggregating (monomerizing) capacity of the co-solutes on stacked chromophores. After washing with saturated solutions of caffeine or o-phenanthroline, the chromatin fluorescence by intercalating fluorochromes is reduced or abolished, which suggests competition effects for intercalative binding modes. These results support the use of caffeine, o-phenanthroline, and tannin in spectroscopic and histochemical studies of dye-stuff interactions with DNA and chromatin.     

Assessment of textile dye remediation using biotic and abiotic agents

The aim of the current work was to assess the removal of direct and reactive dyes using biotic and abiotic agents. Removal of dyes and their derivatives from aqueous solutions was investigated using sugarcane bagasse, sawdust, rice straw, charcoal and fungal biomass as dye removing agents. Seven fungal strains known to have high capacity in removing textile dyes were used. Results of this study indicated that Penicillium commune, P. freii, and P. allii removed 96, 64 and 65%, respectively, of direct violet dye after two hours of incubation. In addition, the use of rice straw was shown to be more efficient in dye removal, than was bagasse or sawdust. Rice straw was effective in removing 72% of direct violet dye within 24 hours. However, with reactive dyes, removal activity was reduced to 27%. Similar trends were recorded with the other tested biotic agents, fast removal of reactive dye was not found after 48 hours of contact time. Results of this study indicate that low-cost, renewable, bioadsorption agents are relatively effective in removing textile dyes from solution.     

Decolourisation of diverse industrial dyes by some Phlebia spp. and their comparison with Phanerochaete chrysosporium

Three species of Phlebia, viz. P. brevispora, P. fascicularia and P. floridensis have been evaluated for their potential to decolourise eight industrial dyes including; reactive yellow, reactive orange, reactive red, rathidol scarlet, coracryl black, coracryl pink, coracryl violet and coracryl red. The cultures used for the present study were pre adapted by growing these on yeast glucose agar medium supplemented with Poly-R 478, a reference dye. The fungal cultures were grown in mineral salts broth and harvested after different incubation periods to obtain their cell free enzyme extracts which were then used to assess their ability to decolourise the above mentioned dyes. The extracts obtained from the cultures grown for six days significantly decolourised the tested dyes. The study revealed Phlebia spp. to be better dye decolourisers than Phanerochaete chrysosporium.     

Probing dimerization and intraprotein fluorescence resonance energy transfer in a far-red fluorescent protein from the sea anemone Heteractis crispa

Proteins from Anthozoa species are homologous to the green fluorescent protein (GFP) from Aequorea victoria but with absorption/emission properties extended to longer wavelengths. HcRed is a far-red fluorescent protein originating from the sea anemone Heteractis crispa with absorption and emission maxima at 590 and 650 nm, respectively. We use ultrasensitive fluorescence spectroscopic methods to demonstrate that HcRed occurs as a dimer in solution and to explore the interaction between chromophores within such a dimer. We show that red chromophores within a dimer interact through a Forster-type fluorescence resonance energy transfer (FRET) mechanism. We present spectroscopic evidence for the presence of a yellow chromophore, an immature form of HcRed. This yellow chromophore is involved in directional FRET with the red chromophore when both types of chromophores are part of one dimer. We show that by combining ensemble and single molecule methods in the investigation of HcRed, we are able to sort out subpopulations of chromophores with different photophysical properties and to understand the mechanism of interaction between such chromophores. This study will help in future quantitative microscopy investigations that use HcRed as a fluorescent marker.     

Studies on the relationship between structure and IgE-binding ability of Parietaria judaica allergen I

The main allergen of Parietaria judaica pollen, Par j I, is a glycopolypeptide with mol. wt about 10,000. It shows a considerable charge heterogeneity which is mostly due to the carbohydrate prosthetic groups, since treatment with trifluoromethanesulfonic acid yielded a deglycosylated protein with 8500 mol. wt that displayed only a few bands on IEF, in a narrow pH-region around 5.0. Deglycosylated Par j I exhibited a specific allergenic activity slightly lower than that of native Par j I; however, no allergenic determinants should be located on the sugar moiety since both native and deglycosylated Par j I inhibited up to a similar extent the binding of specific human IgE to P. judaica-coated wells in ELISA. The decrease of specific allergenic activity following deglycosylation could be ascribed to conformational changes evidenced by CD experiments. On the other hand, fluorescence spectroscopy showed that Par j I bears unidentified yellow-brown chromophores strongly linked to the polypeptide chain. These chromophores were not removed by TFMS treatment. Finally, reduction and alkylation caused the complete loss of allergenic activity, showing that disulphide bridges are essential for the IgE-binding ability of Par j I.     

Adsorptive removal of textile dyes from aqueous solutions by dead fungal biomass

Dead fungal biomass prepared from Phanerochaete chrysosporium and Funalia trogii was tested for their efficiency in removal of textile dyes. The effects of contact time, initial dye concentration, amount of dead biomass and agitation rate on dye removal have been determined. Removal of all dyes required a very short time (60 min). Experimental results show that, P. chrysosporium was more effective than F. trogii . An increase in the amount of dead biomass positively affected of the dye removal. The removal efficiency of different amount of biomass was in order 1 g > 0.5 g > 0.2 g > 0.1 g. The highest removal was obtained at 150-200 rpm. Slightly lower removing activities were found at lower agitation rates. This study showed that it was possible to remove textile dyes by dead biomass of P. chrysosporium .     

Renal handling of phenol red. II. The mechanism of substituted phenolsulphophthalein (PSP) dye transport in rabbit kidney tubules in vitro.

1. The uptake of various substituted phenolsulphophthalein dyes by cortical slices of rabbit kidney has been studied in detail in order to obtain more information on the secretory system for organic anions. 2. The rate of initial uptake of dyes and the accumulation after incubation for 2 hr under aerobic conditions increased in the order: phenol red (PR) greater than bromophenol blue (BPB) greater than bromocresol green (BCG) greater than bromothymol blue (BTB), while the reverse order of uptake was observed under anaerobic conditions. There was no difference between the uptake of BTB under aerobic and anaerobic conditions. 3. The accumulation of dyes under anaerobic conditions could be accounted for by binding to tissue constituents. In comparison with PR (Sheikh, 1972), the substituted dyes were found to interact extensively with the 700 G (cell membranes) and cytosol fractions of renal homogenates. 4. Low concentrations of the substituted dyes efficiently inhibited the accumulation of rho-aminohippurate (PAH). The concentration of dye resulting in 50% inhibition of PAH accumulation (KI) agreed well with concentrations estimated to sustain 50% of maximal dye transport (KM). On this basis the affinity of the dyes for the transport system increases in the order: PR less than BPB less than BCG less than BTB. 5. Probenecid, 2,4-dinitrophenol, PAH, octanoate and succinate affected to a smaller extent the uptake and binding of BPB and BCG by renal tissue than that previously shown for PR (Sheikh, 1972). No inhibitory effect of these substances on the accumulation of BTB by kidney tissue was observed. 6. The binding of PSP dyes by phospholipid vesicles (liposomes) and a representative binding protein, human serum albumin, exhibited close similarity to that of binding by renal tissue. Partition experiments involving octanol-water phases indicated that the hydrophobicity of the dyes increased in the order: PR less than BPB less than BCG less than BTB. 7. The results indicate that BTB, despite its inhibitory potency, is not transported by the organic anion system. BPB and BCG are transported to a lesser extent, and interact more strongly with the transport system than does PR. It is suggested that the substituted dyes by virtue of hydrophobic interaction with the transport system reduce the movement of the mobile part of the transport system.     

P2X antagonists inhibit styryl dye entry into hair cells

The styryl pyridinium dyes, FM1-43 and AM1-43, are fluorescent molecules that can permeate the mechanotransduction channels of hair cells, the sensory receptors of the inner ear. When these dyes are applied to hair cells, they enter the cytoplasm rapidly, resulting in a readily detectable intracellular fluorescence that is often used as a molecular indication of mechanotransduction channel activity. However, such dyes can also permeate the ATP receptor, P2X₂. Therefore, we explored the contribution of P2X receptors to the loading of hair cells with AM1-43. The chick inner ear was found to express P2X receptors and to release ATP, similar to the inner ear of mammals, allowing for the endogenous stimulation of P2X receptors. The involvement of these receptors was evaluated pharmacologically, by exposing the sensory epithelium of the chick inner ear to 5 μM AM1-43 under different experimental conditions and measuring the fluorescence in hair cells after fixation of the tissue. Pre-exposure of the tissue to 5 mM EGTA for 15 min, which should eliminate most of the gating “tip links” of the mechanotransduction channels, deceased fluorescence by only 44%. In contrast, P2X receptor antagonists (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid [PPADS], suramin, 2',3′-O-(2,4,6-trinitrophenyl) ATP [TNP-ATP], and d-tubocurarine) had greater effects on dye loading. PPADS, suramin, and TNP-ATP all decreased intracellular AM1-43 fluorescence in hair cells by at least 69% when applied at a concentration of 100 μM. The difference between d-tubocurarine-treated and control fluorescence was statistically insignificant when d-tubocurarine was applied at a concentration that blocks the mechanotransduction channel (200 μM). At a concentration that also blocks P2X₂ receptors (2 mM), d-tubocurarine decreased dye loading by 72%. From these experiments, it appears that AM1-43 can enter hair cells through endogenously activated P2X receptors. Thus, the contribution of P2X receptors to dye entry should be considered when using styryl pyridinium dyes to detect hair cell mechanotransduction channel activity, especially in the absence of explicit mechanical stimulation of stereocilia.     

Azobenzene‐Incorporated Single‐ and Double‐Stranded Polynorbornenes: Facile Synthesis and Diverse Photoresponsive Property

Double‐stranded polymeric ladderphane using azobenzene as linkers and the single‐stranded polynorbornene with azobenzene pendants are synthesized by the ring‐opening metathesis polymerization using Grubbs ruthenium‐based catalyst. Hydrolysis of these as‐prepared polynorbornenes gives the corresponding isotactic single‐stranded polynorbornene having similar polydispersity and degree of polymerization as those of the original polymers. The photosensitive property of double‐stranded ladderphane indicates significant interactions between adjacent chromophores. These single‐ and double‐stranded polynorbornenes incorporating azobenzene chromophores exhibit distinct photoresponsive performance in the dilute solutions. The rate of photoisomerization for double‐stranded ladderphane is found to be from 6 to 9 times slower than that obtained for single‐stranded polymer. This is ascribed to the differences in structure of polymers and the interaction between azobenzene chromophores in the polymers.     

Paired-wavelength spectral approach to measuring the relative concentrations of two localized chromophores in turbid media: an experimental study

We present an experimental test of a new spectral approach that is aimed at quantifying the relative concentrations of two chromophores that are contained in a defect embedded in a turbid medium. The basic steps of our spectral approach are (a) perform a linear tandem scan of the source and detector across the defect; (b) measure the spectral dependence of the maximum change induced by the defect in the scanned intensity; (c) identify a set of appropriate pairs of wavelengths (lambda1, lambda2) at which such maximum intensity changes are the same; and (d) measure the reduced scattering coefficient spectrum of the background medium. For each wavelength pair (lambda1, lambda2), we obtain a measurement of the relative concentrations of the two chromophores, where the only required parameters are the extinction coefficients of the two chromophores and the ratio of the background scattering coefficients at lambda1 and lambda2. In a mixture of two test chromophores (blue food coloring dye and black India ink) contained in a 0.78-cm diameter cylinder, our spectral approach yielded relative concentrations values that were within 6% of their actual values. Although our paired-wavelength spectral approach is not generally applicable to any pair of chromophores, it is suitable for oxyhemoglobin and deoxyhemoglobin and is thus appropriate for oximetry of localized lesions in biological tissues.     

Multilayer build-up of a reactive polymer with α,ω-functionalized chromophores

Alternating multilayers with up to ten layers have been prepared by sequential reaction of a reactive polymer (poly[(1-methylvinyl isocyanate)-alt-(maleic anhydride)]) with α,ω-functionalized chromophores. For this purpose new chromophores were synthesized varying the length of the hydrophobic spacer. The analysis of the multilayer build-up shows that physisorption is more important than chemisorption for fixation of new polymer layers. Thus, a multilayer build-up is only possible with the shorter, more hydrophilic chromophore and not with the long hydrophobic spacer.     

Relationship of chemical structures of textile dyes on the pre-adaptation medium and the potentialities of their biodegradation by Phanerochaete chrysosporium

Azo dye derivatives of azobenzene constitute the largest group of dyes used in the textile industry and possess recalcitrant chemical groups, such as those of azo and sulphonic acid. Some microorganisms are able to degrade these aromatic compounds. In the present work, decolourisation of culture media containing azo dyes by the ligninolytic fungus Phanerochaete chrysosporium was achieved under nitrogen-limited conditions. The dyes used in the study are derivatives of meta- or para-aminosulphonic or aminobenzoic acids and include in their structures groups such as guaiacol or syringol, which are bioaccessible to the lignin degrading fungus P. chrysosporium. The aim of this study was to pre-adapt the microorganism to the structure of the dyes and to establish the relationships of the chemical structure of the dye present in the pre-adaptation medium with the chemical structure of the dye to be degraded. The azo dye used in the pre-adaptation medium that gave the best overall decolourisation performance was a meta-aminosulphonic acid and guaiacol derivative. The azo dye derivative of a meta-aminobenzoic acid and syringol showed a better performance in the decolourisation assays. Preliminary GC-MS studies indicated the formation of a nitroso substituted catechol metabolite, a precursor of aromatic ring cleavage, which was confirmed to occur by an enzymatic assay. The presence of this type of metabolite allows the establishment of a possible metabolic pathway towards mineralisation.     

Enhanced Photoluminescence of Mesostructured Organosilica Films with a High Density of Fluorescent Chromophores

Synthesis of mesostructured materials containing a high density of organic chromophores in the frameworks is highly desired for applications in unique optical devices using energy transfer. However, a dense accumulation of fluorescent chromophores in solid form generally causes concentration quenching. In this study, dense embedding of the chromophore 1,6‐diphenylpyrene within the framework of mesostructured organosilica films results in efficient blue fluorescence emission with high quantum yields of over 0.75. In contrast to conventional fluorescent films where chromophores are diluted and isolated to suppress concentration quenching, increasing the density of 1,6‐diphenylpyrene moieties within the mesostructured framework leads to the efficient formation of highly emissive excimers. Mesostructured frameworks achieving both high light‐absorptivity and efficient fluorescence emission have great potential in light‐emitting materials, fluorescence sensors, and light‐harvesting components for photoluminescent and photocatalytic systems.     

Regeneration of rhodopsin in frog rod outer segments.

1. Bleaching/regeneration cycles were performed in perfused frog retina while the optical transmittance at suitable wave-lengths was measured continously. Rhodopsin was identified from its spectral absorbance, its photosensitivity and from the kinetics of its regeneration. 2. In the absence of the pigment epithelium regeneration was complete when not more than 2-5% of the rhodopsin initially present had been bleached. However, the cycles could be repeated to a total of regenerated rhodopsin exceeding that explicable on the utilization of stored chromophores. The rate of regeneration was fast, with 0-12 min-1 rate constant, following first order reaction kinetics. Under these conditions the cycle does not seem to involve stages beyond metarhodopsin II. With the moderate bleaching intensities used, half-time 53 min, the Bunsen-Roscoe law was obeyed up to 15 min, indicating a capacity for the photoproducts to be accomondated in situ for subsequent regeneration. 3. It is concluded that only substantial bleaches, which exceed that capacity, result in hydrolysed chromophores. These surplus chromophores become esterified and are temporarily taken up by the pigment epithelium to be re-entered into the visual cycle as fast as they can be processed by the regenerative machinery of the rod outer segments.     

Selective staining of animal chromosomes with synthetic dyes following iodine-dye-procedure.

The paper embodies results of the use of 51 synthetic dyes, belonging to different chemical groups for staining of animal chromosomes following iodine-dye procedure. It has been found that some of these dyes can replace gentian violet, crystal violet and safranin when used after this procedure. It has further been found that the fluorescent dyes, acriflavine and acridine yellow can also be used to stain animal chromosomes and that some of the dyes belonging to one chemical group can be successfully used whereas others of the same group are of no use. Dyes of the monoazo group are absolutely useless. Amongst the dyes successfully used, the preparations remain stable when stained with most of them except methyl green, malachite green, brillant green, iodine green and cresyl violet and amongst acid dyes, acid fuchsin. Cytochemical studies presented herein indicate that the components of the animal chromosomes stainable with crystal violet are the nucleic acids and that these substances should be highly polymerised and should not be even in a semi-degraded state. Removal of any one of these nucleic acids makes the chromosomes unstainable with iodine-crystal violet.     

Reduction in the mutagenicity of synthetic dyes by successive treatment with activated sludge and the ligninolytic fungus, Irpex lacteus

Synthetic dyes are released in wastewater from textile manufacturing plants, and many of these dyes are genotoxic. In the present study, the mutagenicity of azo, anthraquinone, and triphenyl methane dyes was investigated before and after successive biodegradation with activated sludge and the ligninolytic fungus, Irpex lacteus. Two biodegradation systems were used to reduce the genotoxicity of dyes that were not efficiently inactivated by activated sludge alone. Mutagenicity was monitored with the Salmonella reversion assay conducted with the base-pair substitution detector strains, TA100 and YG1042, and the frame-shift detector strains, TA98 and YG1041, with and without rat liver S9. All dyes except for Congo Red (CR) were mutagenic with S9 activation. Assays conducted with the dyes indicated that only the azo dye Reactive Orange 16 (RO16) was mutagenic in both TA98 and TA100. Methyl Red and Disperse Blue 3 (DB3) were mutagenic in TA98, YG1041 and YG1042, while Reactive Black 5 was mutagenic in YG1041 and YG1042. Remazol Brilliant Blue R (RBBR), Crystal violet (CV) and Bromophenol Blue (BPB) were mutagenic only in TA98, but the toxicity of the latter two dyes complicated the evaluation of their mutagenicity. CR was not mutagenic in any of the tester strains. Biodegradation studies conducted with RO16 and DB3 indicated that the two-step biodegradation process reduced the mutagenic potential of RO16 and DB3 to a greater extent than activated sludge alone; the mutagenicity of the two dyes was reduced by 95.2% and 77.8%, respectively, by the two-step process. These data indicate that the combined biodegradation process may be useful for reducing the mutagenicity associated with wastewater from textile factories that contain recalcitrant dyes.