Enzymatic browning in apple products and its inhibition treatments: A comprehensive review.

Apple (Malus domestica) is widely consumed by consumers from various regions. It contains a high number of phenolic compounds (majorly hydroxybenzoic acids, hydroxycinnamic acids, flavanols, flavonols, dihydrochalcones, and anthocyanins) and antioxidant activity, which are beneficial for human health. The trends on healthy and fresh food have driven the food industry to produce minimally processed apple, such as fresh-cut, puree, juice, and so on without degrading the quality of products. Enzymatic browning is one of the problems found in minimally processed apple as it causes the undesirable dark color as well as the degradation of phenolics and antioxidant activity, which then reduces the health benefits of apple. Proper inhibition is needed to maintain the quality of minimally processed apple with minimal changes in sensory properties. This review summarizes the inhibition of enzymatic browning of apple products based on recent studies using the conventional and nonconventional processing, as well as using synthetic and natural antibrowning agents. Nonconventional processing and the use of natural antibrowning agents can be used as promising treatments to prevent enzymatic browning in minimally processed apple products. Combination of 2-3 treatments can improve the effective inhibition of enzymatic browning. Further studies, such on as other potential natural antibrowning agents and their mechanisms of action, should be conducted.

Impacts of microplastics addition on sediment environmental properties, enzymatic activities and bacterial diversity.

Environmental microplastic accumulation can lead to a range of problems and the potential loss of ecosystem services. However, the impacts of microplastics on sediment environmental properties and microbial ecosystems remain unclear. Therefore, this article aimed to better elucidate the effects of microplastics on sediment physicochemical properties, enzymatic activities and the diversity and composition of sediment microbial communities. We conducted a 60-day sediment-incubation experiment using sediments with three concentrations (2%, 5%, and 10%, w/w) and two types (polyethylene (PE) and polyvinyl chloride (PVC)) of microplastics (550 µm) in a climate-controlled chamber. The addition of both PE and PVC microplastics reduced sediment catalase, polyphenol oxidase (PO), and urease activities, and decreased physicochemical indicators, including total organic carbon (TOC), total nitrogen (TN), and pH value. In addition, microplastics reduced bacterial community diversity and inhibited the recovery of community richness, implying that microplastics may compete with sediment microorganisms for niches. Stronger effects were generally detected under a high microplastic concentration (10%), reflecting the influence of the concentration of microplastic exposure on the bacterial community. The dominant bacterial phyla were Chloroflexi, Proteobacteria, Bacteroidota, Firmicutes, and Actinobacteriota, and the relative abundances of Proteobacteria, and Bacteroidota significantly increased in the microplastic treatments during the late incubation period, indicating that microplastic addition may have improved nutritional conditions later in the incubation process. Structural equation modeling indicated that shifts in PO activity had a significant correlation with changes in Proteobacteria abundance (P < 0.01), and important drivers affecting the dissimilarity of Bacteroidota abundance were the changes of TN content and catalase activity (P < 0.05). These results indicated that microplastics with diverse characteristics affected the environmental properties of the sediment, while the physicochemical properties and enzymatic activity of the sediment could directly or indirectly exert different impacts on the dominant bacteria. This study can provide a theoretical basis for the ecological effects of microplastic contamination.

Antagonistic Effect of Plant Growth-Promoting Fungi Against Fusarium Wilt Disease in Tomato: In vitro and In vivo Study.

Fusarium wilt is considered one of the most destructive diseases for tomato plants. The novelty of this work was to investigate the antifungal and plant growth-promoting capabilities of some plant growth-promoting fungi (PGPF). Plant growth-promoting fungi (PGPF) improved the plant health and control plant infections. In this study, two fungal strains as PGPF were isolated and identified as Aspergillus fumigatus and Rhizopus oryzae using molecular method. The extracts of A. fumigatus and R. oryzae exhibited promising antifungal activity against F. oxysporum in vitro. Moreover, antagonistic effect of A. fumigatus and R. oryzae against F. oxysporum causing tomato wilt disease was evaluated in vivo. Disease severity and growth markers were recorded and in vitro antagonistic activity assay of the isolated A. fumigatus and R. oryzae against Fusarium oxysporum was measured. Physiological markers of defense in plant as response to stimulate systemic resistance (SR) were recorded. Our results indicated that A. fumigatus and R. oryzae decreased the percentage of disease severity by 12.5 and 37.5%, respectively. In addition, they exhibited relatively high protection percentage of 86.35 and 59.06% respectively. Fusarium wilt was declined the growth parameters, photosynthetic pigments, total soluble carbohydrate, and total soluble protein, whereas content of free proline, total phenols, and the activity of antioxidant enzymes activity increased under infection. Moreover, application of A. fumigatus and R. oryzae on infected plants successfully recovered the loss of morphological traits, photosynthetic pigment total carbohydrates, and total soluble proteins in comparison to infected control plants. PGPF strains in both non-infected and infected plants showed several responses in number and density of peroxidase (POD) and polyphenol oxidase (PPO) isozymes.

Peroxidase from jackfruit: Purification, characterization and thermal inactivation.

Peroxidase (POD) from jackfruit bulb was purified using ammonium sulfate precipitation, hydrophobic interaction and gel filtration columns. The POD was a dimer with a molecular weight of 104kDa. The Km and Vmax values for guaiacol, gallic acid and o­phenylenediamine (OPD) were estimated. OPD was the most suitable substrate. The enzyme showed its maximum activity at pH5.5 and 55-60°C. The activation energy (Ea) of heat inactivation was estimated to be 206.40kJ/mol. The enthalpy, free energy and entropy values for the thermal inactivation were also determined. The POD activity was enhanced by K+, Zn2+, Ba2+, citric acid, malic acid, benzoic acid and EDTA·Na2, but inhibited by Cu2+, Ca2+, glutathione, cysteine and ascorbic acid. Chemical modification indicated a histidine residue was located in the enzyme active site. The POD activity in fruit extracts significantly decreased when heated at 80°C and 90°C. The ferric-reducing antioxidant power, ABTS radical scavenging activity and total phenolics decreased with increasing heating temperature and time.

Identification, characterization and immune response of prophenoloxidase from the blue swimmer crab Portunus pelagicus and its antibiofilm activity.

Prophenoloxidase is a conserved Cu-containing enzyme acting as a major defense molecule in the immune response of crustaceans. In the present research, we purified prophenoloxidase from the haemolymph of Portunus pelagicus (Pp-proPO) by Blue Sepharose CL-6B chromatography. Pp-proPO exhibited only one band with molecular weight of 75kDa on SDS-PAGE. The purified Pp-proPO was characterized through X-ray diffraction (XRD) and high-performance liquid chromatography (HPLC). Pp-proPO showed phagocytic activity on the yeast Saccharomyces cerevisiae as well as encapsulation on sepharose CL-6B beads associated with CM sepharose and beads of sodium alginate. Pp-proPO also led to strong agglutination on human erythrocytes. Furthermore, Pp-proPO showed magnified PO activity when altered with activated particles acting as pathogen combined molecular patterns (PAMPs), metal ions or other chemicals. Pp-proPO showed relevant antibiofilm activity on Gram negative bacteria Pseudomonas aeruginosa and Escherichia coli. Overall, the above results allowed us to claim that Pp-proPO play a key role in immune defense mechanisms of P. pelagicus crabs, in particular towards microbial pathogens; notably we added basic information to the functional characterization of Pp-proPO, as well as to understand its immunological role in crustaceans defense systems.

Defense-related Proteins from Chelidonium majus L. as Important Components of its Latex.

The aim of this review is to cover most recent research on plant pathogenesis- and defenserelated proteins from latex-bearing medicinal plant Chelidonium majus (Papaveraceae) in the context of its importance for latex activity, function, pharmacological activities, and antiviral medicinal use. These results are compared with other latex-bearing plant species and recent research on proteins and chemical compounds contained in their latex. This is the first review, which clearly summarizes pathogenesisrelated (PR) protein families in latex-bearing plants pointing into their possible functions. The possible antiviral function of the latex by naming the abundant proteins present therein is also emphasized. Finally latex-borne defense system is hypothesized to constitute a novel type of preformed immediate defense response against viral, but also non-viral pathogens, and herbivores.

The effects of PPO activity on the proteome of ingested red clover and implications for improving the nutrition of grazing cattle.

UNLABELLED: Increasing the rumen-stable protein content of feed would lead to improved nitrogen utilisation in cattle, and less nitrogenous waste. Red clover (Trifolium pratense L.) is a high protein ruminant feed containing high polyphenol oxidase (PPO) activity. PPO mediated protein-quinone binding has been linked to protecting plant proteins from proteolysis. To explore the mechanism underlying the effect of PPO on protein protection in fresh forage feeds, proteomic components of feed down-boli produced from wild-type red clover and a low PPO mutant, at point of ingestion and after 4h in vitro incubation with rumen inoculum were analysed. Significant differences in proteomic profiles between wild-type and mutant red clover were determined after 4h incubation, with over 50% less spots in mutant than wild-type proteomes, indicating decreased proteolysis in the latter. Protein identifications revealed preferentially retained proteins localised within the chloroplast, suggesting that PPO mediated protection in the wild-type operates due to the proximity of target proteins to the enzyme and substrates, either diffusing into this compartment from the vacuole or are present in the chloroplast. This increased understanding of protein targets of PPO indicates that wider exploitation of the trait could contribute to increased protein use efficiency in grazing cattle. BIOLOGICAL SIGNIFICANCE: One of the main challenges for sustainable livestock farming is improving capture of dietary nitrogen by ruminants. Typically up to 70% of ingested protein-N is excreted representing a loss of productivity potential and a serious environmental problem in terms of nitrogenous pollution of lands and water. Identification of key characteristics of rumen-protected protein will deliver target traits for selection in forage breeding programmes. The chloroplastic enzyme PPO catalyzes the oxidation of phenols to quinones, which react with protein. Little is currently known about the intracellular protein targets of the products of PPO activity or the mechanism underlying protein complexing, including whether there is any specificity to the reaction. Here we have determined significant differences in the proteomes of freshly ingested down boli corresponding to the presence or absence of active PPO. These results show that in the presence of PPO the forage protein is less amenable to proteolysis and provide the novel information that the protected proteins are putatively chloroplastically located. These data also contribute to a growing evidence base that a chloroplastic PPO substrate exists in red clover in addition to the currently known vacuolar substrates.

Immune indices and identical functions of two prophenoloxidases from the haemolymph of green tiger shrimp Penaeus semisulcatus and its antibiofilm activity.

In the present study, we purified two prophenoloxidases (proPO) from haemolymph of green tiger shrimp, Penaeus semisulcatus by gel fermentation chromatography using blue Sepharose matrix. The two purified prophenoloxidase macromolecules are of about 76 and 75 kDa determined through SDS-PAGE and named as Penaeus semisulcatus prophenoloxidase I (PSproPO I) and Penaeus semisulcatus prophenoloxidase II (PSproPO II). It was further characterized by X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD) and High Performance Liquid Chromatography (HPLC) analysis. The purified PSproPO I and PSproPO II showed the strongest agglutination titre against human erythrocytes compared to goat RBC. The PSproPO I and PSproPO II showed phagocytic activity against yeast Saccharomyces cerevisiae and encapsulation activity against Sepharose CL 6B beads compared to CM Sepharose and Sodium alginate beads. The functional analysis of purified PSproPO I and PSproPO II showed enhanced PO activity when added with the triggering molecules such as pathogen associated molecular patterns (PAMPs), metals and chemicals. In addition, eluted fraction containing PSproPO I and PSproPO II showed antibiofilm activity against Gram positive and Gram negative bacteria. The above results concluded that no significant differences were found between the purified PSproPO I and PSproPO II immune indices and functions. This study might provide a sensitive platform to understand more about the critical roles of PSproPO I and PSproPO II in crustacean immune system.

Targeted water soluble copper-tetrazolate complexes: interactions with biomolecules and catecholase like activities.

Two new mononuclear water soluble copper(II) complexes, [Cu{(5-pyrazinyl)tetrazolate}2(1,10-phenanthroline)] 1 and [Cu{(5-pyrazinyl)tetrazolate}(1,10-phenanthroline)2](NO3)0.5(N3)0.5 2, have been synthesized using the metal mediated [2 + 3] cycloaddition reaction between copper bound azide and pyrazinecarbonitrile. The interactions of these copper tetrazolate complexes 1 and 2 with biomolecules like DNA and bovine serum albumin (BSA) are studied and the catecholase like catalytic activity of compound 2 is also explored. Structural determination reveals that both compounds 1 and 2 are octahedral in nature. Screening tests were conducted to quantify the binding ability of complexes (1 and 2) towards DNA and it was revealed that complex 2 has a stronger affinity to bind to CT-DNA. DFT studies indicated that a lower HOMO-LUMO energy gap between the DNA fragment and metal complexes might be the reason for this type of stronger interaction. DNA cleavage activity was explored by gel-electrophoresis and moderate to strong DNA cleavage properties were observed in the presence and absence of co-reagents. Inhibition of cleavage in the presence of sodium azide indicates the propagation of the activity through the production of singlet molecular oxygen. Furthermore enzyme kinetic studies reflect that complex 2 is also effective in mimicking catecholase like activities. An ESI-MS spectral study indicates the probable involvement of dimeric species [(phen)2Cu-(OH)2-Cu(phen)2](2+) in the catalytic cycle.

Synthesis, structure, and catecholase activity of bispyrazolylacetate copper(II) complexes.

A series of six-coordination copper(ii) complexes containing bis(3,5-di-t-butylpyrazol-1-yl)acetate (bdtbpza) and N-heterocycles or chelating aliphatic ligands have been synthesized. The steric bulkiness of bis(pyrazol-1-yl)acetate anchors two bdtbpza to situate a trans position and to adopt an O-bound monodentate coordination mode with other nitrogen bases occupying the basal plane. Five mononuclear mixed ligand complexes, [Cu(bdtbpza)2(py)4] , [Cu(bdtbpza)2(t-Bupy)4] , [Cu(bdtbpza)2(pym)2(MeOH)2] , [Cu(bdtbpza)2(eda)2] , [Cu(bdtbpza)2(tmeda)(H2O)2] , where py = pyridine, t-Bupy = tert-butylpyridine, pym = pyrimidine, eda = ethylenediamine, and tmeda = tetramethylethylenediamine, were isolated and thoroughly characterized. Intriguingly, the heteroleptic complex , which has two aquo-ligands oriented in the cis positions, demonstrates higher catecholase-like activity in performing aerial oxidation of 3,5-di-tert-butylcatechol (3,5-DTBC) to 3,5-di-tert-butylquinone (3,5-DTBQ) than other bis(pyrazolyl)acetate-embedded copper complexes reported herein, which suggests the essential role of labile cis-aquo ligands to promote the catalytic reaction.

Antioxidant capacity of chewing stick miswak Salvadora persica.

BACKGROUND: Chewing stick (miswak Salvadora persica L.) is an effective tool for oral hygiene. It possessed various biological properties including significant antibacterial and anti-fungal effects. In the present study, we evaluated the antioxidant compounds in miswak. METHOD: Miswak root was extracted with 80% methanol. Methanol extract as antioxidant was evaluated by using DPPH, ABTS and phosphomolybdenum complex assays and analysis by GC-MS. Peroxidase, catalase and polyphenoloxidase assays were performed for crude extract of miswak root. RESULTS: The methanol extract of miswak contained the highest amount of crude extract among the various solvent extracts. The methanol extract showed a concentration dependent scavenging of DPPH and ABTS radicals with IC50 values 4.8 and 1.6 µg crude extract, respectively. The total antioxidant activities, based on the reduction of molybdenum (VI) to molybdenum (V), increased with increasing crude extract content. The correlation coefficients (R2) between total crude extract and DPPH, ABTS scavenging activities and the formation of phosphomolybdenum complex were 0.97, 0.99 and 0.95, respectively. The GC-MS analysis showed that the methanol extract doesn't contain phenolic and flavonoid compounds or under detected limit. After silylation of methanol extract, three compounds namely 2-furancarboxaldehyde-5-(hydroxymethyl), furan-2-carboxylic acid-3-methyl- trimethylsilyl ester and D-erythro-pentofuranose-2-deoxy-1,3,5-tris-O-(trimethylsilyl) were identified by GC-MS analysis. These furan derivatives as they contain hydroxyl groups could be possessed antioxidant activities. The antioxidant enzymes were also detected in the miswak extract with high level of peroxidase and low level of catalase and polyphenoloxidase. CONCLUSIONS: The synergistic actions of antioxidant compounds and antioxidant enzymes make miswak is a good chewing stick for oral hygiene and food purposes.

Purification and characterization of polyphenol oxidase from nettle (Urtica dioica L.) and inhibitory effects of some chemicals on enzyme activity.

Polyphenol oxidase (PPO) of nettle (Urtica dioica L.) was extracted and purified through (NH4)2SO4 precipitation, dialysis, and CM-Sephadex ion-exchange chromatography and was used for its characterization. The PPO showed activity to catechol, 4-methylcatechol, L-3,4-dihydroxyphenylalanine (L-DOPA), L-tyrosine, p-cresol, pyrogallol, catechin and trans-cinnamic acid. For each of these eight substrates, optimum conditions such as pH and temperature were determined and L-tyrosine was found to be one of the most suitable substrates. Optimum pH and temperature were found at pH 4.5 and 30 degrees C respectively and Km and Vmax values were 7.90 x 10(-4) M, and 11290 EU/mL for with L-tyrosine as substrate. The inhibitory effect of several inhibitors, L-cysteine chloride, sodium azide, sodium cyanide, benzoic acid, salicylic acid, L-ascorbic acid, glutathione, thiourea, sodium diethyl dithiocarbamate, beta-mercaptoethanol and sodium metabisulfite were tested. The most effective was found to be sodium diethyl dithiocarbamate which acted as a competitive inhibitor with a Ki value of 1.79 x 10(-9)M. In addition one isoenzyme of PPO was detected by native polacrylamide slab gel electrophoresis.

Purification and characterization of Manduca sexta prophenoloxidase-activating proteinase-1, an enzyme involved in insect immune responses.

Early on, we reported the partial purification of prophenoloxidase-activating proteinase-1 (PAP-1) from the tobacco hornworm, Manduca sexta [Proc. Natl. Acad. Sci. USA 95 (1998) 12220]. PAP-1 requires an auxiliary factor for generating active phenoloxidase (PO) [Insect Biochem. Mol. Biol. 33 (2003) 197; Insect Biochem. Mol. Biol. 34 (2004) 731]. To further characterize their roles in the proteolytic activation of prophenoloxidase (proPO), we purified PAP-1 to near homogeneity by hydroxylapatite, dextran sulfate, gel filtration, and lectin affinity chromatography. With 2.4 x 10(3)-fold purification and 20% yield, we obtained 63 microg PAP-1 from about 120 M. sexta prepupal cuticles (approximately 400 g). The purified glycoprotein (Mr=39,810+/-20; pI=5.6) had the highest amidase activity at pH 8.0 and a low salt concentration. The optimal conditions for proPO activation by PAP-1 and SPHs were: pH 8.0-8.4, PAP:SPH=1.5:1, and 0-10 degrees C for 40-50 min. While PAP-1 and SPHs are reasonably heat stable, PO activity generated after 1h incubation was lower at 20 or 30 degrees C than 0-10 degrees C because activated PO was unstable at a higher temperature. The KMs of PAP-1 toward IEARpNA and proPO were 201+/-18 microM and 16.6+/-3.0 microg/ml, respectively, and the absence of SPHs did not significantly affect KM for the synthetic substrate. PO activity and proPO cleavage were reduced in reaction mixtures containing the same amounts of proPO, PAP-1, and SPHs but increasing concentrations of NaCl. Ionic strength of the reaction buffer may reduce proPO-PAP-SPH interactions, proPO processing, and PO assembly.

Virulence factors of Porphyromonas gingivalis are modified by polyphenol oxidase and asparaginase.

Porphyromonas gingivalis is a well-adapted pathogen of the periodontal pocket distinguished by its wide array of proteolytic activities and its ability to adhere to multiple substrata in the oral cavity. Microbial proteins with binding functions (such as adhesins and enzymes) very often contain critical tyrosine residues, supported by one or more asparagines in the binding cleft. This study investigates the reduction in adhesiveness and in proteolytic activity after treating P. gingivalis with the tyrosine- and asparagine-targeting enzymes polyphenol oxidase (PPO) and asparaginase (ASG). Cysteine protease activity was reduced by pretreatment with both enzymes, while the trypsin-like activity was affected only by PPO. Adhesion to buccal epithelial cells, laminin and fibronectin as well as hemagglutination was reduced by one or both of the enzymes. PPO, but not ASG, reduced the coaggregation of P. gingivalis with Actinomyces naeslundii. Treatment with these enzymes might provide an alternative to traditional antimicrobial strategies.

Combined effect of low-frequency laser and polyphenol oxidase on adhesive activity of Escherichia coli strains isolated from patients with urinary diseases.

The effects of polyphenol oxidase and low-frequency laser irradiation on adhesion of pathogenic Escherichia coli to human erythrocytes and buccal epithelial cells were studied. The maximum decrease in adhesive activity of these strains was observed after complex exposure to laser and enzyme.

Effects of asparaginase and polyphenol oxidase on adhesive characteristics of microorganisms.

We studied the effects of polyphenol oxidase and asparaginase on microorganism adhesion to buccal epithelial cells. These enzymes reduced adhesion of pathogenic microorganisms (uropathogenic and Escherichia coli, Salmonella enteritidis, Entamoeba spp., Influenza virus, Candida albicans, Streptococcus spp.) and had virtually no effect on adhesive characteristics of probiotic variants of Escherichia coli and Lactobacillus fermentum.

Inhibitory effects of plant polyphenoloxidase on colonization factors of Streptococcus sobrinus 6715.

Exogenously added polyphenoloxidase (EC 1.14.18.1), an enzyme which oxidizes tyrosine residues and is commonly found in many dietary components, abolished the aggregation of Streptococcus sobrinus 6715 by high-molecular-weight dextran. The enzyme decreased glucan-binding lectin and/or glucosyltransferase I activities.

The salivary catechol oxidase/peroxidase activities of the mosquito Anopheles albimanus.

Salivary gland homogenates from adult female Anopheles albimanus mosquitoes relaxed aortic rings preconstricted with noradrenaline (NA). This relaxation is slow and is due to destruction of NA. Incubation of NA with the homogenate yielded a product with a spectrum consistent with the corresponding adrenochrome. Oxidation of NA was enhanced by a superoxide generation system and inhibited by the combined action of superoxide dismutase and catalase. Additionally, peroxidase activity on both synthetic (o-dianisidine) and biologically active (serotonin) substrates was also present in the salivary gland homogenates, this latter activity requiring hydrogen peroxide. Noradrenaline oxidation, serotonin and o-dianisidine peroxidation and vasodilation all co-elute with a heme protein of relative molecular mass 50,000, as determined by molecular sieving chromatography. Peroxidase activity was localized in the posterior (female-specific) lobes of salivary glands and was also detected in nitrocellulose membranes probed by hungry mosquitoes. Protein and peroxidase activities were significantly lower in salivary glands of mosquitoes after probing and feeding on blood. It is suggested that adult female Anopheles albimanus mosquitoes contain a salivary heme peroxidase that functions during blood finding and blood feeding by destroying hemostatically active biogenic amines released by the vertebrate host during tissue destruction.

Melanin accelerates the tyrosinase-catalyzed oxygenation of p-hydroxyanisole (MMEH)

Although pigment melanin has long been though of as "inert," recent work has attested to its chemical reactivity. In this communication, we report that either commercial synthetic melanin prepared by persulfate oxidation of tyrosine ("Sigma melanin") or sepia melanin extracted from cuttlefish markedly accelerates the in vitro oxygenation of p-hydroxyanisole (MMEH), catalyzed by mushroom or B-16 melanoma tyrosinase. Kinetics of 4-methoxy-1,2-benzoquinone formation (lambda max = 413 nm) or of molecular O2 uptake were biphasic, with an initial slow rate ("lag time") followed by a fast linear increase. The biphasic response reflects an initial slow hydroxylation followed by a fast dehydrogenation. Added melanin markedly decreased the lag time but had little effect on subsequent dehydrogenation. Similar effects were observed for tyrosine itself. A complex between MMEH and melanin appears to be the "active" species in these reactions. The results indicate that melanin acts as an electron conduit, which accepts electrons from the substrate and transfers them to tyrosinase. The magnitude of the effect depends on the type of melanin as well as on its oxidation state. Kinetic analysis indicates that both melanins are very efficient at transferring electron to tyrosinase, and that Sigma melanin is roughly threefold more efficient than sepia melanin. The qualitative similarity of reaction between the synthetic and "natural" melanins suggests that the former may serve as a first approximation to the in vivo situation. On the other hand, the observed quantitative differences and the sensitivity of these results to the chemical state of melanin suggests that this methodology might eventually be adapted as a non-destructive probe of melanin in situ.(ABSTRACT TRUNCATED AT 250 WORDS)