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Biomedical subjects

G Prota

Publications and source records attributed to G Prota.

At least 55 records · Page 3Linked to original sources
PubMed1993

Inhibitory effect of melanin precursors on arachidonic acid peroxidation.

A possible role of melanin precursors in lipid peroxidation was investigated using the lipoxygenase catalysed oxygenation of arachidonic acid (AA) as a model system. Polarographical monitoring of oxygen consumption showed that, among the metabolites examined, 5,6-dihydroxyindole (DHI) was the most active in inhibiting AA oxygenation catalysed by 15-lipoxygenase. The inhibition was found to be concentration-dependent with an IC50 value of 15 microM. Similar effects were observed in the case of the 5-lipoxygenase promoted reaction. Periodical HPLC analysis of the oxidation mixture showed that, in the presence of DHI, the rate of substrate consumption is markedly reduced. The inhibitory potency was significantly increased either by preincubation of DHI with the enzyme or by increasing the time of residence of the indole in aerated buffer solutions prior to contact with the enzyme. Addition of catalase to the incubation mixture resulted in a partial removal of DHI inhibition. From these and other experiments, an inhibition mechanism is proposed which involves inactivation of the enzyme by reactive species, especially hydrogen peroxide, arising from DHI autoxidation.

Animals↗
PubMed1993

Hair melanins and hair color: ultrastructural and biochemical aspects.

The color variants of mammalian hair, including spotting and albinism, are the result of melanocyte activity and have been shown to be determined by the action of multiple genes, some of which operate through the milieu in which the pigment cell resides; others appear to act intracellularly to control the type of melanogenesis. Although there has been much descriptive work on the mode of action of these genes, it has only been with the recent advances in the chemistry and molecular biology of melanin pigmentation that some progress is being made in understanding the nature and origin of hair color. It is the purpose of this article to provide an integrated overview of the major advances so made and to draw attention to certain peculiarities of the melanization processes of hair with respect to those underlying skin pigmentation. Key words: melanins, melanocytes, melanogenesis, hair.

Hair↗
PubMed1993

Regulatory mechanisms of melanogenesis: beyond the tyrosinase concept.

The last decade has witnessed a quantum jump in our knowledge of the chemistry of melanogenesis and the emergence of some understanding as to how the chemical and enzymatic activities are integrated and regulated at cellular level. To an unusual extent, most of these advances stemmed from a molecular approach involving direct analysis of natural melanins coupled with studies of the chemical reactivity of the putative intermediates under biologically relevant conditions. Thus, bit by bit it has been possible to reconstruct in vitro the overall chemical activity of melanocytes, and to identify a number of regulatory factors other than tyrosinase. This and other approaches, especially the molecular biology of the pigment-related genes, have radically changed and expanded the traditional concept of melanogenesis far beyond the boundaries of the Raper-Mason pathway. Recently, I suggested an improved scheme of melanogenesis accounting for the formation of melanins and related products of melanocyte activity. The present contribution surveys some verifications of the predictions, and certain new break-throughs that have been made in the understanding of the regulatory steps after the dopaquinone stage. It also aims to provide an overview of what is presently known about the structure of the melanin polymers, and to draw attention to some problems pertaining to the definition and classification of the various types of pigments that can be found in epidermal tissues.

Humans↗
PubMed1993

Non-melanosomal regulatory factors in melanogenesis.

The enzyme activity of tyrosinase, catalase, and peroxidase and the number of metal ions in melanogenic subcellular compartments in pigment cells were measured. Coated vesicles were richest in tyrosinase and catalase, whereas premelanosomes had the highest amount of peroxidase. Among metals ions examined, copper, zinc, and iron were more concentrated in pre-melanosomes than in coated vesicles. A quantitative analysis revealed that peroxidase served to enhance eumelanin polymer formation from monomers in the presence of hydrogen peroxide and metal ions, especially copper ions, which had the greatest enhancing effect on the conversion of monomers to polymers.

Animals↗
PubMed1992

5,6-Dihydroxyindole-2-carboxylic acid is incorporated in mammalian melanin.

The role of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) in the biosynthesis of melanins has been studied by using the incorporation of specifically radiolabelled melanogenic precursors into melanins formed by melanocytes growing in vitro and in vivo. Extracts of mouse melanocytes and intact viable melanocytes were found to incorporate into melanin from 25% to more than 60% of [1-14C]tyrosine. Melanins from melanoma tumours grown in mice were radiolabelled with 3,4-dihydroxy[1-14C]phenylalanine, purified and chemoselectively decarboxylated. Determination of the 14CO2 evolved showed that at least 20% of the precursor incorporated in vivo retains the label in the form of non-aminoacidic aromatic-type carboxyl groups. These results provide the first unambiguous demonstration that DHICA is incorporated in physiologically relevant amounts in mammalian melanins.

Animals↗
PubMed1992

Photodynamic degradation of vitamin E induced by psoralens.

Psoralens and other furocoumarins currently used in PUVA photochemotherapy are shown to have, to a variable extent, the ability to hasten the rate of ultraviolet-induced photooxidation of alpha-tocopherol (alpha-T) in ethanol or ethanol-phosphate buffer (pH 6.8). The sensitizing effect varies significantly with the substrate concentration and the nature of the furocoumarin used, and is dependent on the presence of oxygen. Scavengers of singlet oxygen, e.g., sodium azide, markedly inhibit the psoralen-sensitized photooxidation of alpha-T, whereas superoxide dismutase exerts an opposite, accelerating effect on the reaction rate. Catalase has no significant influence on the kinetics of alpha-T decay. Analysis of the products formed by psoralen-sensitized photooxidation of alpha-T in ethanol-phosphate buffer showed the presence of alpha-tocopherolquinone, its 2,3-epoxide and two related compounds containing the 7-oxaspiro[4.5]dec-1-ene-3,6-dione ring system. The nature of these products, coupled with the results of the kinetic experiments, suggest that psoralens induce a type II, oxygen-dependent photodegradation of alpha-T primarily mediated by singlet oxygen.

Chromatography, High Pressure Liquid↗
PubMed1992

Synthesis and cytotoxic properties of new N-substituted 4-aminophenol derivatives with a potential as antimelanoma agents.

New tyrosinase-targeted compounds based on structural variants of the prototype unit 4-aminophenol have been synthesized and screened for their potential as antitumour agents against malignant melanoma. Cytotoxicity assays showed that N-4-hydroxyphenylglycine (NHPG) and its alpha-methyl derivatives methylphenylglycine and dimethylphenylglycine exhibit significant antiproliferative effects on pigmented human melanoma cell lines (HBL), with inhibitory concentrations at 50% (IC50) around 80 micrograms/ml. A marked increase in cytotoxicity was observed with morpholine-containing 4-aminophenols, e.g. N-(2-morpholinoethyl)-4-aminophenol, which showed an IC50 of 20 micrograms/ml of HBL cells. Much more pronounced was the effect of the diacetoxy-derivative, DiAcMoAc, which showed an IC50 of 15 micrograms/ml on HBL cells and as low as 2 micrograms/ml on tyrosinase-containing, non-pigmented human melanoma cells (LND1), with a toxicity response of the same order of magnitude as that of melphalan. These results open interesting perspectives in the design of new targeted pro-drugs against malignant melanoma.

Aminophenols↗
PubMed1991

Comparative action of dopachrome tautomerase and metal ions on the rearrangement of dopachrome.

A vis-a-vis comparison between the effects of dopachrome tautomerase (DCT) and metal ions, e.g., cupric ions, on the kinetics and mode of rearrangement of dopachrome has been carried out under appropriate analytical conditions. The enzyme-promoted reaction is highly stereospecific for L-dopachrome, is unaffected by metal chelators and has an optimal pH around 6.8. By contrast, the kinetics of dopachrome rearrangement catalysed by cupric ions are not dependent on the stereochemistry of the substrate, are affected by EDTA and are not influenced by the pH of the medium in the range between 5-7.5. Both cupric ions and DCT catalyse the rearrangement of dopachrome to give 5,6-dihydroxyindole-2-carboxylic acid (DICA) rather than 5,6-dihydroxyindole (DI). However, at comparable activity, the ratio of formation DICA/DI is significantly higher in the enzyme-catalysed than in the metal-catalysed reaction. These results provide an improved background to look into the mode of action of DCT and metal ions, enabling a clear cut differentiation between the effects of the two factors when both are present in biological extracts.

Animals↗
PubMed1991

2-Aryl-1,3-thiazolidines as masked sulfhydryl agents for inhibition of melanogenesis.

As a part of an ongoing project aimed at developing new skin depigmenting agents, the ability of variously substituted 2-aryl-1,3-thiazolidines to inhibit melanogenesis in vitro was investigated. At 0.2 mM concentration 2-(2'-hydroxyphenyl)-1,3-thiazolidine-4-carboxylic acid (Th2), as well as the descarboxy analog (Th1) and, to a lower extent, the 4'-hydroxy isomer (Th3) all proved capable of preventing the tyrosinase catalyzed conversion of 0.2 mM L-tyrosine to melanin. Spectrophotometric monitoring of the reaction course in the presence of Th2 showed the initial formation of a yellow chromophore (lambda max 400 nm) which slowly decayed, being eventually replaced by a new absorption maximum centered at 305 nm. HPLC analysis of the final incubation mixture revealed the presence of a major product (lambda max 306 nm), ninhydrin and ferric chloride positive, which was isolated by gel filtration on Sephadex G-10 and was identified as beta-[7-(3-carboxy-5-hydroxy-3,4-dihydro-2H-1,4-benzothiazinyl)]al anine (DBA) by 1H-NMR spectroscopy. Attempts to isolate the intermediate with lambda max 400 nm were hampered by its marked instability under the usual chromatographic conditions. However, the nature of the chromophore, coupled with mechanistic considerations, suggested for the compound the Schiff base-containing structure 3,4-dihydroxy-5-S-(N-salicylidenecysteinyl)phenylalanine (salcysdopa). This was substantiated by: (i) the formation of a zinc complex (lambda max 349 nm) analogous to that observed with the model Schiff base N-salicylidene leucine; and (ii) detection by 1H-NMR of a Schiff base resonance at delta 8.1 during the yellow chromophoric phase of the reaction. It was concluded that 1,3-thiazolidines inhibit melanin formation by a mechanism that involves the trapping of enzymically generated dopaquinone by the -SH containing Schiff base arising by cleavage of the thiazolidine ring. The salcysdopa adduct thus formed undergoes hydrolysis and subsequent ring closure to give eventually the colorless DBA.

Alanine↗
PubMed1991

Peroxidase as an alternative to tyrosinase in the oxidative polymerization of 5,6-dihydroxyindoles to melanin(s).

The ability of the peroxidase/H2O2 system to promote the oxidative polymerization of 5,6-dihydroxyindole (DI) and 5,6-dihydroxyindole-2-carboxylic acid (DICA) to melanin pigments was investigated in comparison with tyrosinase. commonly regarded as the sole enzyme involved in melanogenesis. In 0.025 M phosphate buffer at pH 6.8, tyrosinase (2.7 x 10(-3) U/ml) induced a smooth oxidation of 3.0 x 10(-5) M DI (initial rate = 4.4 x 10(-5) M/s) to give a complex mixture of products with the 2,4'-dimer I as the main component, whereas, under the same conditions, peroxidase (0.44 U/ml) and 1.2 x 10(-4) M H2O2 caused the instantaneous conversion of the substrate to a well-defined pattern of products, comprising the 2,4'-and 2,7'-DI dimers I and II, and the related trimers III and IV. When 3.0 x 10(-5) M DICA was used as the substrate, the difference in the effectiveness of the enzymes was much more pronounced. Thus, while peroxidase accomplished the fast oxidation of the indole, yielding the dimer V and the trimer VI as the main products, tyrosinase proved unable to induce more than a poor and sluggish reaction with an initial rate of 5.6.10(-6) M/s. These results raise the possibility that peroxidase, rather than, or in addition to, tyrosinase, may play a critical role in the later stages of the biosynthesis of melanins.

Chromatography, High Pressure Liquid↗
PubMed1991

Mechanism of inhibition of melanogenesis by hydroquinone.

Hydroquinone (HQ) is one of the most effective inhibitors of melanogenesis in vitro and in vivo, and is widely used for the treatment of melanosis and other hyperpigmentary disorders. In an attempt to get some insight into the molecular mechanism of the depigmenting action, which is still very poorly understood, we have investigated the effect of HQ on the tyrosinase catalysed conversion of tyrosine to melanin. Incubation of 0.5 mM tyrosine with 0.07 U/ml tyrosinase in phosphate buffer at pH 6.8 in the presence of 0.5 mM HQ led to no detectable melanin formation, due to the preferential oxidation of HQ with respect to tyrosine (HPLC evidence). Kinetic investigations showed that HQ is a poorer substrate of tyrosinase than tyrosine; yet, it may be effectively oxidised in the presence of tyrosine owing to the generation of catalytic amounts of dopa acting as cofactor of tyrosinase. Product analysis of HQ oxidation with tyrosinase in the presence of dopa showed the predominant formation in the early stages of hydroxybenzoquinone (HBQ), arising from enzymic hydroxylation and subsequent oxidation of HQ, along with lower amounts of benzoquinone (BQ). These results suggest that the depigmenting activity of HQ may partly be related to the ability of the compound to act as an alternate substrate of tyrosinase, thereby competing for tyrosine oxidation in active melanocytes.

Benzoquinones↗
PubMed1991

Role of skin surface lipids in UV-induced epidermal cell changes.

Ultraviolet irradiation is capable of affecting skin surface lipids, especially squalene and cholesterol, both in vitro and in vivo, with generation of active lipoperoxides. The photodecomposition of the skin lipid component was carefully evaluated by capillary gas-chromatography. The effects of UV-induced lipoperoxides on human keratinocytes in culture and on guinea pig ear slices were compared with those of synthetic lipoperoxides, i.e. cumene hydroperoxide and 13-hydroperoxylinoleate. A time- and dose-dependent effect on protein synthesis and mitotic activity was observed. In cell culture low concentrations (0.05-5 micrograms/ml) of peroxidated squalene and synthetic lipoperoxides stimulated the incorporation of radiolabelled thymidine and phenylalanine, while higher concentrations (greater than 10 micrograms/ml), or longer periods of treatment, induced cellular damage. In guinea pig ear slices, the lipoperoxides (5-50 micrograms/ml) increased aminoacid incorporation and the number of epidermal pigment cells; higher concentrations (greater than 100 micrograms/ml) caused a derangement of epidermal structure. The results suggest that UV irradiation of skin generates lipoperoxides from the surface lipids which, in vitro, are capable of producing a number of changes in epidermal cells.

Adult↗
PubMed1990

Selective uptake of 2-thiouracil into melanin-producing systems depends on chemical binding to enzymically generated dopaquinone.

2-Thiouracil (TU), an antithyroid drug, is receiving growing interest as a specific tumor marker for malignant melanoma, owing to its capability of being selectively accumulated into active melanin-producing tissues. However, up until now, the molecular mechanism of TU uptake by growing melanin has remained largely unknown. In an attempt to fill this gap, we have investigated the effect of TU on the tyrosinase catalyzed oxidation of tyrosine. At a concentration of 0.5 mM, TU was found to totally inhibit melanin formation by tyrosinase catalyzed oxidation of 0.25 mM tyrosine in phosphate buffer at pH 6.8. Polarographical monitoring of oxygen consumption under conditions of complete suppression of melanogenesis revealed a significant tyrosinase activity, with TU acting as a modest non-competitive inhibitor of the enzyme (Ki = 0.6 mM). HPLC and TLC analysis of the tyrosine-tyrosinase reaction in the presence of excess TU showed that the substrate is progressively consumed and a major hitherto unknown product (lambda max = 284 nm), positive to ninhydrin and ferric chloride, is concomitantly formed. This was isolated by repeated gel filtration chromatography of the reaction mixture on Sephadex G-10 and was formulated as the TU-dopa adduct 3,4-dihydroxy-6-(4'-hydroxypyrimidinyl-2'-thio)phenylalanine by spectral analysis. These results suggest that selective TU incorporation in pigmented melanomas and other melanin-producing systems is due to the covalent binding to dopaquinone, produced by tyrosinase catalyzed oxidation of tyrosine.

Animals↗
PubMed1990

Activation of mammalian tyrosinase by ferrous ions.

Kinetic experiments are reported showing that mammalian tyrosinase from B16 mouse melanoma is significantly activated by catalytic amounts of ferrous ions. Monitoring of tyrosine oxidation by both dopachrome formation and oxygen consumption showed that ferrous ions at micromolar concentrations induce a marked enzymatic activity with 0.01 U/ml of highly purified tyrosinase, whereas no detectable reaction occurs in the absence of metal over a sufficiently prolonged period of time. The extent of the activating effect, which is specific for the reduced form of iron, is proportional to the concentration of the added metal with a typical saturation profile, no further effect being observed beyond a threshold value. Changing the buffer system from phosphate to hepes or tris results in a marked decrease of the Fe2(+)-induced activation. Scavengers of active oxygen species, such as superoxide dismutase, catalase, formate and mannitol have no detectable effect on the tyrosinase activity. These results are accounted for in terms of an activation mechanism involving reduction of the cupric ions at the active site of the resting enzyme.

Animals↗
PubMed1990

Decrease of total, glutathione and cysteine SH in non-alcoholic cirrhosis.

The content of plasma thiols was determined in 42 patients with non-alcoholic cirrhosis. Total thiols were evaluated by Ellman's method, glutathione and cysteine by HPLC in fluorescence. Cirrhotic patients showed a significant decrease in plasma thiol content with respect to a control group of 32 healthy subjects, as total sulphydryls as well as glutathione and cysteine. The thiol decrease does not seem to be related to nutritional status or dietetic factors. Our data indicate that liver cirrhosis, independently from alcohol abuse, produces a decrease in all plasma thiols, probably secondary to a complex alteration of the transsulfuration pathway.

Adult↗