Melanin-like Pigment Research Articles

Albino Strains of Ophiostoma Species for Biological Control of Sapstaining Fungi

SummaryAlbino strains ofOphiostoma floccosum,O. piceaeandO. pluriannulatumwere selected and screened for biological control of sapstaining fungi on New Zealand radiata pine (Pinus radiata). Albino strains were obtained through matings and single ascospore isolations from cultures of prevalent species in New Zealand. These strains do not synthesize the melanin-like hyphal pigments of common sapstaining fungi. Additional mating studies were also carried out to obtain isolates ofO. piceaeandO. floccosumthat lacked pigmented fruiting structures. Several albino isolates ofO. piceaewith colorless synnemata and isolates ofO. floccosumwith little to no synnemata were obtained. Biological control potential of the albino isolates was evaluated in the laboratory by challenging them on wood chips with fungi that cause extensive sapstain in pine,Leptographium procerum,Ophiostoma piliferumandSphaeropsis sapinea. Many albino isolates ofO. floccosum,O. piceaeandO. pluriannulatumwere effective in stopping the challenge fungi from staining wood chips and were fast growing and colorless when grown unchallenged on wood chips. Selected albino isolates ofO. pluriannulatumwere used in two field trials in New Zealand to control sapstain. Several strains were found to significantly reduce dark sapstain as compared to the untreated control logs.

Micro-pixe study on sorption behaviors of cobalt by lichen biomass

Sorption behavior of Cobalt (Co) by lichen biomass has been studied in the foliose lichen Permotrema tinctorum in solution between pH 3 and 5. Disc of P. tinctorum, weighing ≈9 mg was incubated in 25 ml 0.1 M NaCl solutions containing 1 mM Co for up to 96 h. Concentrations of Co in the solutions were measured at 24, 48, 72 and 96 h by ICP-AES. Spatial distribution of Co, K, P and Fe in the cross-section of P. tinctorum was examined by in-air μ-PIXE (particle induced X-ray emission) technique developed in the TIARA facility of JAERI. Sorption of Co by P. tinctorum reached equilibrium within 24 h. Co uptake was averaged to be 27 dry wt.%, and was independent of solution pHs between 3 and 5. Two-dimensional distribution of Co indicates that the sorbed Co was localized, and the density of Co in the lichen was ordered lower surface > upper surface, medullary layer > algal layer. Highest density in the two-dimensional distribution of Co is obtained in the lower surface suggesting that Co was sorbed by melanin-like pigment contained in the lower surface. Two-dimensional distribution of Co did not correspond to Fe indicating that Co was not adsorbed on the Fe-containing minerals trapped on P. tinctorum. It is concluded that μ-PIXE analysis is a useful technique to detect the distribution of low concentration of elements in lichen biomass.

The liver-protecting activity of melanin-like pigment derived from black tea

The liver-protecting activity of melanin-like pigment (MLP) derived from tea was studied using hydrazine intoxication. The non-reduced MLP demonstrated the lack of activity. Reduction of MLP caused an increase in the number of phenol groups. Administration of reduced MLP (rMLP) to rats (i.p., 20 mg/kg, 30 min before hydrazine) to rats prevented development of heavy liver intoxication. Pretreatment of animals with rMLP prevented the rise of serum alanine transferase activity and the decrease of glutathione level in the liver challenged by hydrazine. It also lowered the hepatic malondialdehyde concentration. Reduced MLP significantly restored the ability for rats to metabolize antipyrine. The introduction of MLP alone did not affect pharmacokinetics of antipyrine nor introduce toxicity to rats. Biochemical and pharmacokinetical data proved that the rMLP protects the liver against hydrazine-induced injury.

Involvement of 5-hydroxytryptophan in melanogenesis.

Tyrosine is generally considered to be the physiological precursor of melanins and tyrosinase the enzyme responsible. However, recent studies have shown that also peroxidases are involved in the biosynthesis of melanins. These enzymes use hydrogen peroxide to oxidise various phenol substrates. In this paper, we used a substrate other than tyrosine, i.e. 5-hydroxytryptophan, to verify if its peroxidase/H2O2-mediated oxidation gave rise to the formation of melanin. We also subjected 5-hydroxytryptophan to the action of tyrosinase, for comparison purposes. We observed that both enzymes converted this substrate to melanin and that peroxidase, in the presence of hydrogen peroxide, was much more effective than tyrosinase in catalysing the oxidative polymerization of 5-hydroxytryptophan, with the formation of insoluble black melanin-like pigments. Samples deriving from the reaction-substrate enzyme were ultrafiltered at different times through an Amicon ultrafiltration cell equipped with an Amicon Diaflo XM-50 membrane, in order to remove the enzyme, and immediately lyophilised. The resulting samples were analysed by matrix assisted laser desorption/ionisation (MALDI) mass spectrometry, which clearly identified several oligomer species in the reaction mixture. This work was undertaken to investigate the possible precursors of neuromelanin and the enzyme responsible for melanogenesis in brain, since although the central nervous system does not contain tyrosinase, it is rich in peroxidase and hydrogen peroxide.

Melanization of Cryptococcus neoformans and Histoplasma capsulatum reduces their susceptibilities to amphotericin B and caspofungin.

The fungal pathogens Cryptococcus neoformans and Histoplasma capsulatum produce melanin-like pigments in the presence of L-dopa in vitro and during mammalian infection. We investigated whether melanization affected the susceptibilities of the fungi to amphotericin B, caspofungin, fluconazole, itraconazole, or flucytosine (5FC). Using the standard macrodilution MIC protocol (the M27A protocol) of the National Committee for Clinical Laboratory Standards for yeast, we found no difference in the susceptibilities of melanized and nonmelanized C. neoformans and H. capsulatum isolates. Killing assays demonstrated that melanization reduced the susceptibilities of both fungi to amphotericin B and caspofungin. Laccase-deficient C. neoformans cells grown with L-dopa were significantly more susceptible than congenic melanin-producing yeast to killing by amphotericin B or caspofungin. Preincubation of amphotericin B or caspofungin with melanins decreased their antifungal activities. Elemental analysis of melanins incubated with amphotericin B or caspofungin revealed an alteration in the C:N ratios of the melanins, which indicated binding of these drugs by the melanins. In contrast, incubation of fluconazole, itraconazole, or 5FC with melanins did not significantly affect the antifungal efficacies of the drugs or the chemical composition of the melanins. The results suggest a potential explanation for the inefficacy of caspofungin against C. neoformans in vivo, despite activity in vitro. Furthermore, the results indicate that fungal melanins protect C. neoformans and H. capsulatum from the activities of amphotericin B and caspofungin and that this protection is not demonstrable by standard broth macrodilution assays.

Histoplasma capsulatum synthesizes melanin-like pigments in vitro and during mammalian infection.

Melanin is made by several important pathogenic fungi and has been implicated in the pathogenesis of a number of fungal infections. This study investigated whether the thermally dimorphic fungal pathogen Histoplasma capsulatum var. capsulatum produced melanin or melanin-like compounds in vitro and during infection. Growth of H. capsulatum mycelia in chemically defined minimal medium produced pigmented conidia. Growth of H. capsulatum yeast in chemically defined minimal medium with L-3,4-dihydroxyphenylalanine (DOPA) or (-)-epinephrine produced pigmented cells. Treatment of the pigmented cells with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were similar in size and shape to their respective propagules. Melanin-binding monoclonal antibodies (MAb) labeled pigmented conidia, yeast, and the isolated particles as determined by immunofluorescence microscopy. Electron spin resonance spectroscopy revealed that pigmented yeast cells and particles derived from pigmented cells were stable free radicals consistent with their identification as melanins. Tissues from mice infected with H. capsulatum and from biopsy specimens from a patient with histoplasmosis contained fungal cells that were labeled by melanin-binding MAb. Digestion of infected mouse tissues yielded dark particles that reacted with the melanin-binding MAb and were similar in appearance to H. capsulatum yeast cells. Additionally, sera from infected mice contained antibodies that bound melanin particles. Phenoloxidase activity capable of synthesizing melanin from L-DOPA was detected in cytoplasmic yeast cell extracts. These findings indicate that H. capsulatum conidia and yeast can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role to play in the pathogenesis of histoplasmosis.

A case of Dubin-Johnson Syndrome

Dubin-Johnson syndrome is a form of benign, familial idiopathic jaundice presenting with chronic intermittent conjugated hyperbilirubinemia and a melanin-like pigment has been found in the parenchymal liver cells. This disorder is rarely diagnosed in the neonatal period. We report a case of Dubin-Johnson syndrome presenting with neonatal cholestasis.

The larger cup fungi in Britain: part 4: Sarcoscyphaceae and Sarcosomataceae

The first article in this series (Spooner, 2000) provided an introduction to cup fungi and noted that most of the larger species belong to the order Pezizales. These are commonly known as the ‘operculate cup-fungi’ based on their ascus structure – mostly thinwalled and dehiscing by an apical ‘lid’ or operculum and include an estimated 300 or so species in Britain. A key to the 13 families of Pezizales represented in Britain was provided, and two subsequent articles (Spooner 2001a, 2001b) provided keys for the identification of the British species of the family Pezizaceae. In the present article two further families, Sarcoscyphaceae and Sarcosomataceae, are considered. These families are closely related to each other, and indeed some authors have preferred not to separate them but to merge all their included species in a single family Sarcoscyphaceae. They differ significantly from other Pezizales most notably in ascus and spore characters. The asci, always non-amyloid, are rather thick-walled, with the operculum usually subapical, positioned slightly to one side of the apex – often termed ‘suboperculate’. In addition, the spores of these fungi are commonly asymmetrical, often curved or inequilateral, unlike those of almost all other Pezizales.They are also unusual in being multinucleate, otherwise characteristic in the Pezizales only of Morchellaceae. Under the microscope the ascus and spore characters are usually fairly easy to observe, but many of the species that belong in this group can be so recognised either by their particularly brightly coloured apothecia (Sarcoscyphaceae) or their dull, brown to blackish colours (Sarcosomataceae). The bright, usually red or orange colours are due to the presence of abundant carotenoid pigments, these usually being absent in the dark-coloured species in which melanin-like pigments occur instead.The apothecia of these fungi vary greatly in size and shape, some being small, sessile, discoid structures others being large, cupulate, often stalked and sometimes even compound.They occur mostly in spring and are saprotrophs, occurring on the ground or on rotting wood. Although Sarcoscyphaceae exhibits its greatest diversity in the tropics, both of these families are virtually cosmopolitan in distribution. However, they include between them no more than about 60 species in all and, in Britain, just nine species ranged in six genera have been recorded. Furthermore, it should be noted that some of the British species are rare and, regrettably, two of them, the beautiful and distinctive Microstoma protracta, and the obscure Pithya vulgaris, both known here from ancient records from only single localities, are surely now extinct in Britain. A key plus brief descriptions for the British genera and species are provided in this account.

Optimization of high molecular weight pullulan production by Aureobasidium pullulans in batch fermentations.

Of five strains of Aureobasidium pullulans studied, NRRL Y-2311-1 yielded the highest titer (26.2 g/L) of pullulan and formed the lowest amount of melanin-like pigment. Sucrose was superior to glucose as the carbon and energy source on the basis of yield and titer of pullulan produced. Pullulan titer was higher (26.2 vs 5.1 g/L), biomass concentration was lower (6.9 vs 12.7 g/L), and DO was lower (0 vs 60% of saturation) when the fermenter was agitated by a marine propeller compared to Rushton impellers. Pullulan produced by strain NRRL Y-2311-1 ranged in weight-average molar mass (M(w)) from 486 KDa and number-average molar mass (M(n)) from 220 Da on day 1 of growth to 390 KDa and 690 Da on day 6; M(w) declined by about 35% from day 1 to day 3, the day of maximum pullulan titer. For the other strains, the ranges of molar mass on the day of maximum pullulan titer were 338-614 KDa (M(w)) and 100-6820 Da (M(n)).

Protective activity of melanin-like pigment derived from tea on Drosophila melasnogaster against the toxic effects of benzidine

The protective activity of melanin-like pigment (MLP) derived from tea was studied using benzidine (BZ) as a DNA-reactive chemical. A wild strain of Drosophila melanogaster, Canton-S, was used to evaluate the toxic effect of BZ and protective power of MLP. Addition of MLP to feed at concentrations of 1.5–15 μg/ml decreased the toxic effect of BZ and increased the survival of the flies of both sexes. By monitoring chemiluminescence intensity, it was found that MLP prevented the production of free radicals due to metabolic transformation of BZ. MLP also prevented the formation of DNA–DNA cross-links and 8-hydroxy-deoxyguanosine (8-OH-dG) DNA adducts. BZ can form free radial intermediates that bind to DNA sites, blocking the expression of genes responsible for the flies’ resistance to harmful factors. MLP exhibited antioxidant activity by binding to these intermediates.

The Lly protein is essential for p-hydroxyphenylpyruvate dioxygenase activity in Legionella pneumophila

The lly locus confers fluorescence, haemolysis, brown pigmentation and an increased resistance to light in Legionella pneumophila. In this study, we correlated the pigment production of two lly-positive L. pneumophila isolates and a recombinant lly-positive Escherichia coli strain with the presence of homogentisic acid (HGA) in the culture supernatant. The detection of HGA by high performance liquid chromatography and the data analysis of the deduced amino acid sequence of the lly gene indicate that the lly locus codes for a p-hydroxyphenylpyruvate dioxygenase (HPPD). This enzyme catalyses the transformation of p-hydroxyphenylpyruvate into HGA, which subsequently oxidises and polymerises into a melanin-like pigment. One open reading frame (ORF 1) in the lly region exhibited homologies with genes of Synechocystis sp., Petroselium crispum and Streptomyces mycarofaciens that code for methyltransferases. By screening a genomic library of L. pneumophila (serogroup 1) strain Corby with a monoclonal antibody against the legiolysin ( lly), we identified two recombinant E. coli clones that did not produce the brown pigment and showed no haemolysis and fluorescence. DNA sequencing revealed that both clones contained 874 nucleotides of the N-terminal part of the lly gene. The recombinant strains expressed truncated legiolysin proteins of 39.5 and 35.7 kDa and did not produce HGA. Considering the highly conserved structure of legiolysin-like HPPD genes from other organisms, we suggest that the C-terminus of the legiolysin may be essential for the enzymatic activity that conferred pigmentation via HGA polymerisation, haemolysis and fluorescence.

Detection of melanin-like pigments in the dimorphic fungal pathogen Paracoccidioides brasiliensis in vitro and during infection.

Melanins are implicated in the pathogenesis of several human diseases, including some microbial infections. In this study, we analyzed whether the conidia and the yeasts of the thermally dimorphic fungal pathogen Paracoccidioides brasiliensis produce melanin or melanin-like compounds in vitro and during infection. Growth of P. brasiliensis mycelia on water agar alone produced pigmented conidia, and growth of yeasts in minimal medium with L-3,4-dihydroxyphenylalanine (L-DOPA) produced pigmented cells. Digestion of the pigmented conidia and yeasts with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were the same size and shape as their propagules. Immunofluorescence analysis demonstrated reactivity of a melanin-binding monoclonal antibody (MAb) with the pigmented conidia, yeasts, and particles. Electron spin resonance spectroscopy identified the yeast-derived particles produced in vitro when P. brasiliensis was grown in L-DOPA medium as a melanin-like compound. Nonreducing polyacrylamide gel electrophoresis of cytoplasmic yeast extract revealed a protein that catalyzed melanin synthesis from L-DOPA. The melanin binding MAb reacted with yeast cells in tissue from mice infected with P. brasiliensis. Finally digestion of infected tissue liberated particles reactive to the melanin binding MAb that had the typical morphology of P. brasiliensis yeasts. These data strongly suggest that P. brasiliensis propagules, both conidia and yeast cells, can produce melanin or melanin-like compounds in vitro and in vivo. Based on what is known about the function of melanin in the virulence of other fungi, this pigment may play a role in the pathogenesis of paracoccidioidomycosis.

A novel melanin-like pigment derived from black tea leaves with immuno-stimulating activity

Melanin-like pigment (MLP) was isolated from black tea leaves by alkaline extraction, acid hydrolysis, and repeated precipitation, and a yield of 2% (dry weight basis) was obtained. The isolated MLP possessed physical and chemical properties comparable to melanins extracted from other sources. The immuno-modulating activity of MLP was measured using plaque-forming cells assay. Oral administration of MLP significantly stimulated the spleen lymphoid tissue in mice at doses ranging from 50 to 200 mg/kg body weight, with a maximal antibody response at 75 mg/kg. The immuno-stimulating activity of tea MLP may serve as a platform for drug discovery, and the abundance of MLP from tea suggests an economical source for health food application.

Autofluorescence of melanins induced by ultraviolet radiation and near ultraviolet light. A histochemical and biochemical study.

The induction of autofluorescence of melanins by UV radiation (330-380 nm) and near UV (400-440 nm) light (jointly called UV light) was studied in tissue sections using three commercially available mounting media. Only Immu-Mount (Shandon) was found suitable for this purpose. UV irradiation of melanins in sections mounted in this medium induced strong yellow autofluorescence irrespective of the type of the polymer (eumelanin, neuromelanin, pheomelanin and ochronotic pigment). The phenomenon of autofluorescence induction was also observed with isolated natural and in vitro prepared melanins. It was inhibited by anhydrous conditions, sodium azide and catalase. In parallel experiments, rapid degradation of melanins with an intermediate fluorescent stage was achieved in UV-irradiated sections mounted in media artificially enriched with hydrogen peroxide, or directly in aqueous solutions of H2O2, Na2O2 or HIO4. Oxidations not associated with UV light led to nonfluorogenic breakdown of melanins. These observations indicate that the common mechanism may be an oxidative attack resulting from a concerted action of hydrogen peroxide and UV light leading, through strongly fluorescent intermediates, to a complete bleaching and oxidative breakdown of melanin and melanin-like polymers. Reactive oxygen species (including ozone) are considered to be important reactants in these experiments. Lipopigments differ from melanin-like pigments by their primary autofluorescence, which mostly faded during continuous prolonged irradiation. The only regular exception was melanosis coli pigment, the autofluorescence of which was considerably augmented by UV irradiation. Our results demonstrate a novel type of fluorogen in autofluorescent pigment histochemistry. The implications of the results are discussed especially in the light of the possible presence of melanin-based fluorogens in lipopigments.

Melanization ofCryptococcus neoformansreduces its susceptibility to the antimicrobial effects of silver nitrate

Cryptococcus neoformans is a human pathogenic fungus that is frequently found in avian feces and Eucalyptus trees. There is evidence that C. neoformans can make a melanin-like pigment in pigeon excreta, a major natural environmental niche. Silver nitrate, AgNO3, is a highly toxic compound for bacteria and fungi. In this study we investigated the effects of melanin production by C. neoformans on the susceptibility of this fungus to AgNO3. C. neoformans was grown in media with and without the melanin precursor, L-dopa, for various times and susceptibility to AgNO3 was determined by measuring percentage of survival after incubation in AgNO3. There was an inverse association between time allowed for melanization and susceptibility to Ag+. Addition of melanin particles to a suspension of non-melanized C. neoformans cells reduced their susceptibility to AgNO3, consistent with metal ion chelation by melanin. Binding of Ag+ to melanin particles was demonstrated by atomic absorption spectroscopy. The results indicate that melanization of C. neoformans reduces susceptibility to a toxic heavy metal. This suggests a role for melanin in environmental protection against heavy metal toxicity.

Toxic effects of apomorphine on rat cultured neurons and glial C6 cells, and protection with antioxidants

Many catechol derivatives are currently used as drugs, even if they produce reactive oxygen species that may cause tissue damage. Among them, apomorphine, a potent dopamine agonist, displays efficient anti-parkinsonian properties, but the consequences of its oxidant and toxic properties have been poorly investigated on in vitro models. In the present work, we investigated apomorphine cytotoxicity by incubating cultures of rat glioma C6 cells and primary cultures of neurons with different concentrations of the drug. Apomorphine-promoted cell death was proportional to its concentration and was time-dependent. The ed 50 of apomorphine on C6 cell death after 48 hr was about 200 μM. The cytotoxic effects induced by apomorphine were correlated to its autoxidation, which leads to the formation of reactive oxygen species, semiquinones, quinones, and a melanin-like pigment. C6 cells that underwent treatment with 400 μM apomorphine for 6 hr displayed features of necrosis, including loss of membrane integrity, degeneration of mitochondria, and DNA fragmentation. Thiols, such as cysteine, N-acetyl- l-cysteine, and glutathione, significantly protected cultured neurons and C6 cells against apomorphine-induced cytotoxicity. Thiols also inhibited apomorphine autoxidation. These data strongly suggest that apomorphine cytotoxicity towards neurons and C6 cells results from an intracellular oxidative stress.

Mechanism of the incidental production of a melanin-like pigment during 6-demethylchlortetracycline production in Streptomyces aureofaciens.

The secondary metabolite 6-demethylchlortetracycline (6-DCT), which is produced by Streptomyces aureofaciens, is used as a precursor of semisynthetic tetracyclines. Strains that produce 6-DCT also produce a melanin-like pigment (MP). The correlation between MP production and 6-DCT production was investigated by using S. aureofaciens NRRL 3203. Production of both MP and 6-DCT was repressed by phosphate or ammonium ions, suggesting that syntheses of these compounds are controlled by the same regulators. Ten chlortetracycline-producing recombinants were derived from 6-DCT-producing mutant NRRL 3203 by gene replacement. All of the recombinants produced chlortetracycline but not MP, indicating that MP production is the results of a defect in the 6-methylation step and suggesting that the polyketide nonaketideamide is a common intermediate leading to MP as well as 6-DCT. To further examine the possibility that MP might be synthesized via the 6-DCT-biosynthetic pathway, mutants defective in 6-DCT biosynthesis were derived from a 6-DCT producer. Some of these mutants were able to produce MP, while others, including mutants with mutations in the gene encoding anhydrotetracycline oxygenase, an enzyme catalyzing the penultimate step in the pathway, produced neither 6-DCT nor MP. Production of 6-DCT and production of MP were restored simultaneously by integrative transformation with the corresponding 6-DCT-biosynthetic genes, indicating that some of 6-DCT-biosynthetic enzymes are indispensable for MP production. These findings suggest that a defect in the 6-methylation step results in redirection of carbon flux from a certain intermediate in the 6-DCT-biosynthetic pathway to a shunt pathway and results in MP production.

Black Thyroid Adenoma

A 22-year-old woman with a "hot" thyroid nodule who was being treated with minocycline for severe acne vulgaris is presented. A partial thyroidectomy specimen revealed a black adenoma. Microscopically, the black pigment was found in the follicular cells and the colloid of the adenoma. The pigment was bleached with potassium permanganate and was Fontana-Masson stain positive. It was negative for iron, periodic acid-Schiff, and acid-fast Ziehl-Neelsen stains. All these feature suggest a melanin-like pigment. However, electron microscopic examination revealed a dense osmophilic material present within the lysosomes of the follicular cells. No melanosomes were present. Twenty-six previously reported cases are reviewed, and the possible mechanisms for the deposition of the pigment in the adenoma and its relation to minocycline degradation products are discussed.

Nitrite- and peroxide-dependent oxidation pathways of dopamine: 6-nitrodopamine and 6-hydroxydopamine formation as potential contributory mechanisms of oxidative stress- and nitric oxide-induced neurotoxicity in neuronal degeneration.

In the presence of nitrite ions (NO(2)(-)) in phosphate buffer (pH 7. 4) and at 37 degrees C, dopamine was oxidized by a variety of hydrogen peroxide (H(2)O(2))-dependent enzymatic and chemical systems to give, in addition to black melanin-like pigments via 5, 6-dihydroxyindoles, small amounts of the potent neurotoxin 6-hydroxydopamine (1) and of 6-nitrodopamine (2), a putative reaction product of dopamine with NO-derived species. Treatment of 0. 5 or 1 mM dopamine with horseradish peroxidase (HRP) or lactoperoxidase (LPO) in the presence of 1 or 2 mM H(2)O(2) with NO(2)(-) at a concentration of 0.5-10 mM resulted in the formation of 1 and 2 in up to 8 and 2 microM yields, respectively, depending on the substrate concentration and the NO(2)(-):H(2)O(2) ratio. Nitration and hydroxylation of 0.1 mM dopamine was observed with 1 mM NO(2)(-) using HRP and the D-glucose/glucose oxidase system to generate H(2)O(2) in situ. In the presence of NO(2)(-)-, Fe(2+)-, or Fe(2+)/EDTA-promoted oxidations of dopamine with H(2)O(2) also led to the formation of 1 and 2, the apparent product ratios varying with peroxide concentration and the partitioning of the metal between EDTA and catecholamine chelates. In the presence of NO(2)(-), Fe(2+)-promoted autoxidation of dopamine gave 2 but no detectable 1. When injected into the brains of laboratory rats, 2 caused sporadic behavioral changes, indicating that it could elicit a neurotoxic response, albeit to a lower extent than 1. Model experiments using tyrosinase as an oxidizing system and mechanistic considerations suggested that formation of 2 does not involve reactive nitrogen radicals but results mainly from nucleophilic attack of NO(2)(-) to dopamine quinone. Generation of 1, on the other hand, may be derives from different H(2)O(2)-dependent pathways. Collectively, these results outline a complex interplay of NO(2)(-)- and peroxide-dependent oxidation pathways of dopamine, which may contribute to impair dopaminergic neurotransmission and induce cytotoxic processes in neurodegenerative disorders.

Identification of a novel nutrient-deprivation-induced Sinorhizobium meliloti gene (hmgA) involved in the degradation of tyrosine.

Sinorhizobium meliloti strain N4 carries a Tn5luxAB insertion in a gene which is induced by nitrogen and carbon deprivation as well as in the presence of tyrosine. The Tn5luxAB-tagged locus was found to share significant similarity with the human hmgA gene and the corresponding Aspergillus nidulans gene, encoding the enzyme homogentisate dioxygenase, which is involved in the degradation of tyrosine. Extended DNA sequence analysis of the tagged locus revealed the presence of several ORFs, including one encoding a polypeptide sharing a high degree of similarity with human and fungal maleylacetoacetate isomerases. Strain N4 was found to be unable to use tyrosine as carbon source, to lack homogentisate dioxygenase activity, to produce a melanin-like pigment and to be affected in stationary-phase survival. This is believed to be the first report of a hmgA-homologous gene in bacteria.