Dario Botti

Possible genotoxicity of melanin synthesis intermediates: Tyrosinase reaction products interact with DNA in vitro

SummaryThe actual cellular target of the cytotoxic intermediates of melanin synthesis is not yet known. In the present paper it is shown that eukaryotic DNA binds in vitro to soluble reaction products of tyrosinase (EC 1.14.18.1) and is physically modified, as ascertained by the following criteria: (a) buoyant density in cesium chloride density gradients; (b) polyacrylamide gel electrophoresis: (c) deoxyribonuclease (EC 3.1.4.5) test; (d) electron microscopy. The results reported here support the view that DNA itself may be a target for the cytotoxic intermediates of melanin synthesis.

Tyrosinase-like activity in normal human substantia nigra

A tyrosinase-like activity was found in human substantia nigra by polyacrylamide gel electrophoresis of fractions prepared from homogenates of the substantia nigra. The enzyme activity was detected by staining the gels with L-3,4-dihydroxyphenylalanine, dopamine and 5,6-dihydroxyindole as substrates for tyrosinase (EC 1.14.18.1). A case of parkinsonism does not show the L-3,4-dihydroxyphenylalanine and dopamine oxidase activities.

Avian Cytokines - An Overview

In recent years the knowledge of avian cytokines has advanced and new data are continuously added. Nevertheless, some discontinuities persist and the correlations between molecular and functional levels are not completely clear. Most of the studies are focused on chicken, and comparative aspects with other avian groups are limited. The existence of T1 and T3 avian cytokines was assessed long ago and the recent relevant demonstration of the existence of T2 cytokines in birds is a further step in depicting a more complete view on avian immunology. The progressive knowledge of avian cytokines can hopefully help in developing new strategies in prophylaxis and therapy of avian diseases, not always completely controlled due to the emergence of more pathogenic strains.

Truffle tyrosinase: Properties and activity

Abstract The present paper investigates the l -3,4-dihydroxyphenylalanine oxidase (EC 1.14.18.1) and l -tyrosine 3-monooxygenase (EC 1.14.18.1) activities of truffles of the genus Tuber , a highly pigmented group of Ascomycetes. The laccase (EC 1.10.2.1) activity has also been explored in the homogenate supernatants from these mushrooms. The effects of various inhibitors of tyrosinase, of buffer concentration, temperature and pH on the tyrosinase activity of truffle cytosols have been investigated. The K m values of l -3,4-dihydroxyphenylalanine and l -tyrosine have been calculated and are in the range of those found in other mushrooms (i.e. 0.37 mM and 2.70 mM respectively).The polyacryamide gel electrophoretic pattern of the l -3,4-dihydroxyphenylalanine oxidase activities of different species of truffles have been obtained. Moreover, the truffle pigment formation and localization have been histochemically investigated and correlated with the reproductive differentiation.

Harding-Passey mouse-melanoma tyrosinase inactivation by reaction products and activation by l-epinephrine

1. Harding-Passey mouse-melanoma tyrosinase (EC 1.14.18.1) is inhibited during L-3,4-dihydroxyphenylalanine oxidation by reaction products. L-3,4-dihydroxyphenyl 3-[14C]alanine oxidation products bind to the enzyme, as demonstrated by gel electrophoresis and radioactivity measurements. 2. The enzyme interacts with indoles and oxidizes dopamine and norepinephrine. 3. L-epinephrine activates tyrosinase at non hormonal concentrations and bovine serum albumin protects the enzyme from auto-inhibition. 4. The inhibition of the Harding-Passey mouse-melanoma tyrosinase, during substrate oxidation, is very similar to that of mushroom enzyme.

Inhibition of tyrosinase by indole compounds and reaction products. Protection by albumin.

Tyrosinase activity decreases as the reaction proceeds and is inhibited by L-3,4-dihydroxyphenylalanine oxidation products. Indole and tryptophan inhibit tyrosinase reaction and bovine albumin protects against end-product(s) inhibition or inactivation. Since the same tyrosinase reaction products are indole compounds and some authors reported the binding of indole derivatives with albumin, it is here suggested that indole intermediates of melanin synthesis inhibit or inactivate tyrosinase.

Proteolytic activity of bovine lactoferrin

Bovine lactoferrin catalyzes the hydrolysis of synthetic substrates (i.e., Z-aminoacyl-7-amido-4-methylcoumarin). Values of Km and kcat for the bovine lactoferrin catalyzed hydrolysis of Z-Phe-Arg-7-amido-4-methylcoumarin are 50 μM and 0.03 s−1, respectively, the optimum pH value is 7.5 at 25 °C. The bovine lactoferrin substrate specificity is similar to that of trypsin, while the hydrolysis rate is several orders of magnitude lower than that of trypsin. The bovine lactoferrin catalytic activity is irreversibly inhibited by the serine-protease inhibitors PMSF and Pefabloc. Moreover, both iron-saturation of the protein and LPS addition strongly inhibit the bovine lactoferrin activity. Interestingly, bovine lactoferrin undergoes partial auto-proteolytic cleavage at positions Arg415-Lys 416 and Lys440-Lys441. pKa shift calculations indicate that several Ser residues of bovine lactoferrin display the high nucleophilicity required to potentially catalyze substrate cleavage. However, a definitive identification of the active site awaits further studies.

5,6-Dihydroxyindole oxidation by mammalian, mushroom and amphibian tyrosinase preparations

The oxidation of 5,6-dihydroxyindole by tyrosinases from mushroom, Harding-Passey melanoma, bovine eye and Bufo bufo embryo has been investigated. The apparent Km values for this substrate were measured and found to be of the same order of magnitude as those for L-tyrosine and L-3,4-dihydroxyphenylalanine, as reported in the literature (5 x 10(-4) M). The 5,6-dihydroxyindole oxidases of mushroom and T4 melanoma isozyme are sensitive to phenylthiourea, while, on the other hand, those from crude preparations of bovine and B. bufo tyrosinases are not sensitive to the inhibitor in an evident manner. The action of some indole derivatives on the 5,6-dihydroxyindole oxidase of mushroom has also been investigated.

Partial structures of truffle melanins

Quinonoid and polyphenolic biopolymers are described as the most important constituents of truffle pigments. Both alkali-soluble and insoluble fractions of peridium and gleba tissues of truffles of the genus Tuber have been investigated. All the data collected suggest that these black pigments are allomelanins of polyketide origin.

EFFECTS OF MELANOPROTEINS AND THEIR PRECURSORS ON CELL PROLIFERATION THE PROBLEM OF SELECTIVITY

The actions of melanin and its precursors on mitotic frequencies, cell division and 3H‐thymidine incorporation in protokaryotic and eukaryotic cells are studied. It was also suggested that the binding of melanin precursors with proteins in the melanosomes is a way of scavenging a cytotoxia activity.