Biosorption of copper by fungal melanin

Summary Melanin obtained from Aureobasidium pullulans and Cladosporium resinae was an efficient biosorbent for copper. Copper uptake could be expressed using various adsorption isotherms; melanin from A. pullulans obeyed Freundlich and Langmuir isotherms whereas C. resinae melanin followed the BET isotherm indicating a more complex type of adsorption than in A. pullulans. In general, uptake capacities of melanin were greater than for intact biomass and the higher uptake by pigmented rather than albino biomass could be correlated with the presence of melanin. Cu²⁺ was less readily desorbed from melanin by dilute mineral acids than from intact biomass and again, the relative ease of Cu²⁺ desorption from pre-loaded pigmented or albino biomass was correlated with the presence or absence of melanin. Mg²⁺ and Zn²⁺ appeared to be the most effective cations for desorption with Na⁺ and K⁺ the least effective. The addition of melanin to a coppercontaining culture of the albino strain of A. pullulans resulted in some reduction of toxicity.     

Fungal melanin inhibitor and related compounds from Penicillium decumbens

In Silene vulgaris (M.) G. cell culture three growth phases were distinguished, namely, a lag phase, an exponential phase and a stationary phase. Pectin termed silenan and an acidic arabinogalactan were isolated as cell wall polysaccharides of S. vulgaris callus at the different growth phases during culture. Production of silenan as the galacturonan (or rhamnogalacturonan) core was observed at the beginning of the exponential phase and at the stationary phase of the callus growth. Arabinogalactan, containing the galacturonic acid residues, is formed at the exponential phase followed by attachment to the core of silenan in the middle of the exponential phase. The arabinogalactan constituent of silenan appeared to be destroyed gradually at the stationary growth phase. The monosaccharide compositions of silenan and arabinogalactan were determined at various phases of the callus growth. Silenan was found to be formed in maximum amounts at the exponential phase of the cell growth. Insignificant alterations of the yields of acidic arabinogalactan were found during culture while total productivity per litre of medium and rate of production per day of arabinogalactan were found to be maximal at the exponential phase of growth.     

Ultrastructural localization of heavy metals by a modified sulfide-silver method

Summary An ultrastructural modification and application of the sulfide-silver method for cell suspensions and ultrathin sections of tissue has been described using glutaraldehyde saturated with hydrogen sulfide as a fixative. When, after physical development in a modified sulfide-silver developer, silver grains appeared in fully treated cells and sections but neither in ungassed but developed controls, nor in gassed but non-developed sections, they were thought to give the sites of heavy metals. A positive reaction for heavy metals has been described to occur in the secretion granules of pancreatic -cells of dog, cat, Chinese hamster, rabbit, and rat, as well as the fish Cottus quadricornis. On isolated cells a positive reaction was obtained in the fungus Cryptococcus neoformans, on the surface of all rat peritoneal cells and cattle blood leucocytes, as well as in erythrocytes and in the specific granules of rat mast cells and rat and cattle eosinophils.The limitations and critical methodological points of the procedure are given, as well as the precautions needed in the interpretation of the results.This work has been supported by grants from the Swedish Medical Research Council (project No. 12X-718-01) and from the Medical Faculty, the University of Umeå.     

Oxidation of NADH by melanin: effect of UV light and copper ions.

The oxidation reaction of NADH with synthetic melanin from L-dopa was investigated under various physicochemical conditions both by spectrophotometric measurements and ESR spectroscopy. The different amounts of NADH oxidized and the effects on the formation and decay of melanin free radicals were compared. Some hypotheses on a common physical mechanism involved in electron transfer reaction and in electron excitation through a liquid-solid interface are presented.     

Solution structure of copper ion-induced molecular aggregates of tyrosine melanin.

Melanin, the ubiquitous biological pigment, provides photoprotection by efficient filtration of light and also by its antioxidant behavior. In solutions of synthetic melanin, both optical and antioxidant behavior are affected by the aggregation states of melanin. We have utilized small-angle x-ray and neutron scattering to determine the molecular dimensions of synthetic tyrosine melanin in its unaggregated state in D(2)O and H(2)O to study the structure of melanin aggregates formed in the presence of copper ions at various copper-to-melanin molar ratios. In the absence of copper ions, or at low copper ion concentrations, tyrosine melanin is present in solution as a sheet-like particle with a mean thickness of 12.5 A and a lateral extent of approximately 54 A. At a copper-to-melanin molar ratio of 0.6, melanin aggregates to form long, rod-like structures with a radius of 32 A. At a higher copper ion concentration, with a copper-to-melanin ratio of 1.0, these rod-like structures further aggregate, forming sheet-like structures with a mean thickness of 51 A. A change in the charge of the ionizable groups induced by the addition of copper ions is proposed to account for part of the aggregation. The data also support a model for the copper-induced aggregation of melanin driven by pi stacking assisted by peripheral Cu(2+) complexation. The relationship between our results and a previous hypothesis for reduced cellular damage from bound-to-melanin redox metal ions is also discussed.     

On the use of diethyldithiocarbamate for detection of electron-transferring copper proteins in bacterial respiratory chains

Abstract In a previous study on the anaerobic electron transport in the wild-type and a mutant strain of Paracoccus denitrificans , the differences in sensitivity towards the copper-chelating agent diethyldithiocarbamate have been interpreted to arise from a substitution of a soluble copper protein for the absent cytochrome c ₅₅₀. Here it is shown that two factors complicate this interpretation: (i) diethyldithiocarbamate can also inhibit through interaction with (a) site(s) located in the cytoplasmic membrane and (ii) it has the potential of being a good electron donor for c -type cytochromes. The participation of a copper protein in the denitrification pathway thus needs to be verified by independent means.     

Fungal interactions reduce carbon use efficiency

The efficiency by which fungi decompose organic matter contributes to the amount of carbon that is retained in biomass vs. lost to the atmosphere as respiration. This carbon use efficiency (CUE) is affected by various abiotic conditions, including temperature and nutrient availability. Theoretically, the physiological costs of interspecific interactions should likewise alter CUE, yet the magnitude of these costs is untested. Here we conduct a microcosm experiment to quantify how interactions among wood‐decay basidiomycete fungi alter growth, respiration and CUE across a temperature and nitrogen gradient. We show that species interactions induced consistent declines in CUE, regardless of abiotic conditions. Multispecies communities exhibited reductions in CUE of up to 25% relative to individual CUE, with this biotic effect being greater than the observed variation attributable to abiotic conditions. Our results suggest that the extent to which fungal‐mediated carbon fluxes respond to environmental change may be influenced strongly by species interactions.     

Mammalian melanocytes do not use phenylalanine for melanin synthesis

Hamster melanoma cells (RPMI 3460) were examined for their ability to utilize phenylalanine for melanin biosynthesis. There was a small but significant incorporation of L-[1-1414C] phenylalanine into hot acid-insoluble cellular material in the presence of cycloheximide. However, this radioactivity was removable from the acid-insoluble fraction by pronase digestion. A similar percentage of L-[U-14C] leucine incorporation was likewise resistant to cycloheximide inhibition. Residual protein synthesis is apparently responsible for the incorporation of both amino acids. Cycloheximide did not inhibit melanin synthesis. These results suggest that mammalian melanocytes do not use phenylalanine for melanin synthesis. Phenylalanine is not incorporated directly into melanin, nor do the cells appear to convert it to tyrosine via a phenylalanine hydroxylase.     

Inhibition by melanin of lipid photo-oxidation

Removal of melanosomes from retinal pigment epithelium cells is accompanied by a sharp rise in the rate of VIS light-induced lipid peroxidation. The synthetic DOPA-melanin effectively suppresses the UV light-induced lipid peroxidation of cardiolipin not only through a passive decrease of irradiation (optical screening), but also by active chemical inhibition of the reaction. It is assumed that the observed active inhibition is due to the interaction between DOPA-melanin and the free radical products generated in cardiolipin upon UV illumination. It is concluded that the high photoresistance of melanin-containing cells of retinal pigment epithelium is due to the ability of melanosomes to exert strong inhibition of photo-induced lipid peroxidation.     

Fungal contamination and mycotoxin detection of powdered herbal drugs

Forty-nine powdered herbal drugs were analyzed for their mold profile and for the potential presence of three mycotoxins (aflatoxin, sterigmatocystin, ochratoxin A). Aspergillus and Penicillium species were predominant, but Rhizopus, Mucor, Cladosporium, and Aureobasidium spp. were also found in a few samples. Mycotoxins were not detected in any samples, and only one isolated culture was found to be a mycotoxin producer on laboratory media.     

Inhibition of transglutaminase by synthetic tyrosine melanin

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. Previously, we found a high molecular mass transglutaminase-inhibitory substance produced by Streptomyces lavendulae Y-200 that appeared to be a melanin substance. Here, we report that synthetic tyrosine melanin inhibited various types of transglutaminases. Tyrosine melanin inhibited tissue-type transglutaminase in a competitive manner with a glutamine substrate, and also inhibited the cross-linking of casein catalyzed by a tissue-type transglutaminase. The melanized hemolymph of the silkworm and melanin solutions prepared from melanin precursors inhibited tissue-type transglutaminase. These results suggested that the melanin substances generally inhibit transglutaminases.     

Degradation of melanin by Aspergillus fumigatus.

A strain of Aspergillus fumigatus from composted coffee and garden wastes utilized natural deproteinized insect, banana, hair, octopus, and synthetic tyrosine and dopa melanins as sole sources of carbon. With a sucrose supplement, degradation was essentially complete after 50 days in Czapek medium pH 6.5 at 30 degrees C. The catabolic rate differed for each substrate pigment, as did the molecular weight distribution of products accumulating in the medium. After incubation with L-[U-14C]melanin, over 50% was recovered in a dark fungal pigment, the remainder appearing as cell protein, chitin, lipid, CO2, and polar metabolites. When grown on melanin, the normally pale mycelia darkened with the production of a fungal allomelanin, with infrared spectrum and alkali fusion products differing from those of the substrate pigment. Isotope distribution in amino acids for A. fumigatus grown on labeled melanin supplemented with sucrose suggested separate pools for synthesis of cell proteins and melanoproteins. Deposition of allomelanin increased resistance of conidia, sterigma, and conidiophores to lytic carbohydrases as judged by scanning electron microscopy.     

Waterborne pathogen detection by use of oligonucleotide-based microarrays

A small-oligonucleotide microarray prototype was designed with probes specific for the universal 16S rRNA and cpn60 genes of several pathogens that are usually encountered in wastewaters. In addition to these two targets, wecE-specific oligonucleotide probes were included in the microarray to enhance its discriminating power within the Enterobacteriaceae family. Universal PCR primers were used to amplify variable regions of 16S rRNA, cpn60, and wecE genes directly in Escherichia coli and Salmonella enterica serovar Typhimurium genomic DNA mixtures (binary); E. coli, S. enterica serovar Typhimurium, and Yersinia enterocolitica genomic DNA mixtures (ternary); or wastewater total DNA. Amplified products were fluorescently labeled and hybridized on the prototype chip. The detection sensitivity for S. enterica serovar Typhimurium was estimated to be on the order of 0.1% (10(4) S. enterica genomes) of the total DNA for the combination of PCR followed by microarray hybridization. The sensitivity of the prototype could be increased by hybridizing amplicons generated by PCR targeting genes specific for a bacterial subgroup, such as wecE genes, instead of universal taxonomic amplicons. However, there was evidence of PCR bias affecting the detection limits of a given pathogen as increasing amounts of a different pathogen were spiked into the test samples. These results demonstrate the feasibility of using DNA microarrays in the detection of waterborne pathogens within mixed populations but also raise the problem of PCR bias in such experiments.     

Permeabilization of Fungal Membranes by Plant Defensins Inhibits Fungal Growth

We used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in fungi. Membrane permeabilization induced by plant defensins in Neurospora crassa was biphasic, depending on the plant defensin dose. At high defensin levels (10 to 40 μM), strong permeabilization was detected that could be strongly suppressed by cations in the medium. This permeabilization appears to rely on direct peptide-phospholipid interactions. At lower defensin levels (0.1 to 1 μM), a weaker, but more cation-resistant, permeabilization occurred at concentrations that correlated with the inhibition of fungal growth. Rs-AFP2(Y38G), an inactive variant of the plant defensin Rs-AFP2 from Raphanus sativus, failed to induce cation-resistant permeabilization in N. crassa. Dm-AMP1, a plant defensin from Dahlia merckii, induced cation-resistant membrane permeabilization in yeast (Saccharomyces cerevisiae) which correlated with its antifungal activity. However, Dm-AMP1 could not induce cation-resistant permeabilization in the Dm-AMP1-resistant S. cerevisiae mutant DM1, which has a drastically reduced capacity for binding Dm-AMP1. We think that cation-resistant permeabilization is binding site mediated and linked to the primary cause of fungal growth inhibition induced by plant defensins.     

Fungal contamination and mycotoxin detection of powdered herbal drugs.

Forty-nine powdered herbal drugs were analyzed for their mold profile and for the potential presence of three mycotoxins (aflatoxin, sterigmatocystin, ochratoxin A). Aspergillus and Penicillium species were predominant, but Rhizopus, Mucor, Cladosporium, and Aureobasidium spp. were also found in a few samples. Mycotoxins were not detected in any samples, and only one isolated culture was found to be a mycotoxin producer on laboratory media.     

The formation of glyoxylate from glycine by melanin

Natural and synthetic melanins catalyze the conversion of glycine to glyoxylate and formic acid in vitro. The conversion depends upon the concentration of melanin.