Anna Palumbo

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.

Adrenalin oxidation revisited. New products beyond the adrenochrome stage

Abstract In neutral aqueous buffer adrenochrome ( 1 ), the first isolable intermediate in the oxidation of adrenalin, undergoes rearrangement to give, besides adrenolutin ( 2 ), a yellow compound which was assigned the dimeric structure 6 . Under anaerobic conditions, compound 6 is the major reaction product (about 60 % yield). In the presence of air a more complex pattern of products is formed including, besides 6 , the hitherto unknown 5,6-dihydroxy-l-methyl-2, 3-indoledione ( 10 ) and a related compound identified as the 4,4-dimer 11 .

Occurrence and properties of tyrosinase in the ejected ink of cephalopods

Abstract 1. 1. In addition to melanin, the ejected ink of cephalopods ( Octopus vulgaris, Sepia officinalis and Loligo vulgaris ) was found to contain substantial amounts of active tyrosinase(s). 2. 2. Contrary to early observations little or no enzymic activity was found associated with melanin granules. 3. 3. Despite differences in molecular size and electrophoretic mobilities, the kinetic properties of the cephalopod enzymes paralleled those of tyrosinases from other sources except for a marked preference for the d forms of tyrosine and dopa substrates. 4. 4. A possible physiological role of tyrosinase in the defence mechanism of cephalopod ink is discussed.

Stochastic approaches for sensors placement against intentional contaminations in water distribution systems

Water distribution systems are vulnerable to intentional contamination: in this paper, optimising the placement of a set of monitoring stations to promptly detect this type of attack is considered. Due to the uncertainty about the place and the time of the beginning of the attack, and the inherent variability of the hydraulic conditions throughout the water distribution network, the impact probability distribution (IPD) has been defined in order to take into account the random variability of the conventional measures of impact. Two different approaches for the optimal placement of the monitoring stations are compared: the first minimises the expected value of a conventional damage measure, while the second minimises a given percentile of the IPD. The two approaches are applied to a real-world case study, showing their feasibility.

Optimal design of urban drainage networks

In this paper, a general procedure for the optimal design of urban drainage networks is proposed taking into account realistic technical constraints and the management cost of the automatic flushing devices. The procedure, which is based on the utilisation of a standard genetic algorithm, is first tested with reference to a urban drainage network taken from the literature, while a second test case is considered in order to evaluate the impact of different input parameters and constraints on the optimisation problem. In particular, we evaluate: (i) the effect of considering the crown elevation of the network outlet not fixed a priori but variable in a given range; (ii) the effect of imposing that the size of the conduit downstream a node should be not smaller than the sizes of the conduits upstream; and (iii) the effect of modifying input parameters such as the peak wastewater discharge or the management unit costs.

Optimal Design of Rural Drainage Networks

Abstract In this paper, a novel procedure is presented and demonstrated for the optimal design of rural drainage networks, based on the coupling of a genetic algorithm with suitable hydrologic and hydraulic models. The models, which allow the evaluation of water depth and discharge through the network, differ for the levels of simplification introduced into the representation of physical phenomena (rainfall-runoff transformation and flood wave propagation through channels). Their applications are shown and the use of oversimplified approaches is discussed: although very popular in practice, they can adversely affect the characteristics of the optimal network.

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.

5-thiolhistidine, a new amino acid from eggs of Octopus vulgaris

Abstract 1. 1. Eggs of Octopus vulgaris were found to contain a new amino acid, 5-thiolhistidine, which was isolated and characterized as the corresponding disulphide. 2. 2. Eggs of Loligo vulgaris contain a related metabolite, identified as 1-methyl-5-thiol-histidine. The same amino acid was also detected in urine of Octopus and Laligo . 3. 3. The biochemical interest of these amino acids is briefly discussed in relation to the structure and biogenesis of adenochrome, an unusual iron(III) chelating peptide from Octopus vulgaris .

Isolation of a possible biosynthetic precursor of adenochrome from the white bodies of Octopus vulgaris

Abstract 1. 1. Adenochrome, the red iron-containing pigment found originally in the branchial hearts of Octopus vulgaris, was isolated in the form of desferriadenochrome (DFA) from gills, hepatopancreas, ovaries after egg deposition, and amoebocytes of the animal. 2. 2. Methods for identification of DFA included u.v. spectroscopy, paper electrophoresis and acid degradation. 3. 3. The white bodies were found to contain a more acid DFA-analogue (DFA-WB), identified as DFA-Asp2, which, by partial hydrolysis, was readily converted to authentic DFA by loss of the two aspartic acid residues. 4. 4. A possible role of white bodies and amoebocytes in the biosynthesis and circulation of adenochrome in Octopus vulgaris is discussed.

Isolation and characterization of adenochrome, a unique iron(III)-binding peptide from Octopus vulgaris

Adenochrome, the iron(III)-containing pigment from the branchial heart of Octopus vulgaris, has been isolated as colourless desferri-adenochrome (DFA) and shown to consist of a mixture of closely related peptides derived from glycine and three isomeric amino-acids, adenochromines A, B, and C, which were formulated as the novel dopa derivatives (1a), (1b), and (1c), respectively, by spectral and chemical evidence.