Protective effect of ferulic acid ethyl ester against oxidative stress mediated by UVB irradiation in human epidermal melanocytes.

UV solar radiation is the major environmental risk factor for malignant melanoma. A great effort is currently posed on the search of new compounds able to prevent or reduce UV-mediated cell damage. Ferulic acid is a natural compound recently included in the formulation of solar protecting dermatological products. The purpose of the present work was to assess whether its ethyl ester derivative, FAEE, could protect skin melanocytes from UV-induced oxidative stress and cell damage. Experiments on human melanocytes irradiated with UVB showed that FAEE treatment reduced the generation of ROS, with a net decrease of protein oxidation. FAEE treatment was accompanied by an induction of HSP70 and heme oxygenase, by a marked suppression of PARP activation and a significant suppression of apoptosis. Moreover FAEE prevented iNOS induction, thus suppressing the secondary generation of NO-derived oxidizing agents. FAEE may represent a potentially effective pharmacological approach to reduce UV radiation-induced skin damage.

Interaction of enkephalin derivatives with reactive oxygen species.

The oxidation of opioid peptides by tyrosinase in the presence of an excess of a thiol gives rise to cysteinyldopa derivatives. The major products arising from the reaction between Leu-enkephalin and cysteine are represented by 5-S-cysteinyldopaenkephalin (5-CDenk) and 2-S-cysteinyldopaenkephalin (2-CDenk). The interaction of 5-CDenk and 2-CDenk with reactive oxygen species (ROS) has been studied. These compounds are able to scavenge superoxide anion, hydroxyl and peroxyl radicals as well as to reduce the lipid peroxidation rate induced by ABAP. The scavenging activities in all instances are dose-dependent. In some cases CDenks are more active than compounds recognized as strong radical scavengers, such as Trolox and mannitol. As a result of the action of the Fenton system, the CDenks (as well as the Enks) are oxidized into pigmented derivatives. The possible implications of the interaction of CDenks and Enks with ROS on melanization process in Parkinson's disease are discussed.

Formation of homovanillic acid dimer by enzymatic or Fenton system - catalyzed oxidation.

Homovanillic acid is the most extensively employed reagent for the fluorometric detection of peroxidase. However, the assays based on the determination of the oxidation product of homovanillic acid do not allow a selective detection of the enzyme, because chemical or physical factors can interfere with the fluorometric determination. The aim of this work was to verify if other enzymatic or non-enzymatic systems might catalyze the homovanillic acid oxidation. The reaction was investigated by spectrophotometric and fluorometric assays; HPLC analysis was used to separate homovanillic acid from its oxidation product and to obtain information on the oxidation process. The results obtained showed that soybean lipoxygenase in the presence of hydrogen peroxide can oxidize homovanillic acid with the formation, by an o,o'-biphenyl linkage, of the corresponding dimer as the sole reaction product. The reaction followed Michaelis-Menten kinetics, for both homovanillic acid and hydrogen peroxide. Other systems, such as cytochrome c/H(2)O(2) and Fenton reagents, were also able to oxidize homovanillic acid to its dimer. It can be affirmed that possible interference by other oxidative systems - that could be present in the biological materials tested - should be considered in assays of peroxidase activity based on the detection of the dimer of homovanillic acid.

Cysteinyldopaenkephalins: synthesis, characterization and binding to bovine brain opioid receptors.

The reaction of opioid peptides with mushroom tyrosinase in the presence of an excess of a thiol compound gives rise to cysteinyldopaenkephalins (CDEnks). The major product is represented by the 5-S-CDEnk (80%) and the minor one by the isomer 2-S-CDEnk (20%). The adducts between leucine-enkephalin (Leu-enk) and cysteine have been isolated by high performance liquid chromatography (HPLC) and identified by amino acid analysis and electrospray ion mass spectrometry. 5-S-CDEnk is able to bind to opioid receptors in bovine brain membranes. Its binding affinity is higher for delta than for mu receptors and about 8-fold lesser than that exploited by Leu-enk. In the presence of the peroxidase/H(2)O(2) system, CDEnks can be converted into the corresponding pheo-opiomelanins.

Lipoxygenase/H2O2-catalyzed oxidation of dihdroxyindoles: synthesis of melanin pigments and study of their antioxidant properties.

5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), which are important intermediates in melanogenesis, can be converted into the corresponding melanin pigments by the action of the lipoxygenase/H2O2 system. Kinetic and HPLC analyses indicate that both DHI and DHICA are good substrates for this enzymatic system. Enzyme activity on both substrates was measured in comparison with peroxidase and tyrosinase; the oxidizing behaviour of lipoxygenase is more similar to that of peroxidase rather than that of tyrosinase. The antioxidant properties of DHI- and DHICA-melanins have been investigated in comparison with other kinds of melanins. DHICA-melanin shows a more pronounced antioxidant effect than that of DHI-melanin and this behaviour can be ascribed to the different structure and solubility of the two pigments. The mixed polymer synthesized from DHI and DHICA is the most effective one. Some implications about the possible explanation of the above mentioned behaviour are discussed.

Production of melanin pigments by cytochrome c/H2O2 system.

In the presence of hydrogen peroxide cytochrome c can perform the oxidation of catecholamines and their S-cysteinyl-derivatives yielding melanins as final products. The initial reaction rate is linearly dependent on cytochrome c and H2O2 concentration; the reaction follows the Michaelis and Menten kinetics both for H2O2 and hydrogen donors. Sulfhydryl compounds inhibit the formation of the pigment. The reported data indicate that a heme-containing protein belonging to the mitochondrial chain can accelerate the oxidation of catecholamines to eumelanins.

Melanins from tetrahydroisoquinolines: spectroscopic characteristics, scavenging activity and redox transfer properties.

Tetrahydroisoquinolines (TIQs) are endogenous compounds deriving from the nonenzymatic Pictet-Spengler condensation of catecholamines (CA) with aldehydes. TIQs have been extensively studied in the last years not only because they have been found in the brain of postmortem specimens of Parkinson's patients, but also because they are able to induce parkinsonian symptoms if injected in animals. In the present article we demonstrate that TIQs bearing a catecholic moiety (tetrahydropapaveroline, salsolinol, laudanosoline, and apomorphine) are easily oxidized in the presence of hydrogen peroxide by various enzymes--i.e., peroxidase (POD), lipoxygenase (LOX), and xanthine oxidase (XO)--into the corresponding TIQ-melanins. The kinetic parameters of the above-mentioned reactions and some spectroscopic characteristics of the synthetized pigments are reported. In particular, UV-VIS and EPR spectra emerge as very similar to those exhibited by dopa-melanin. Furthermore, TIQ-melanins appear to be similar to dopa-melanin regarding some specific physico-chemical properties: NADH-oxidizing properties, oxy-radicals scavenging activity, and ability to form soluble mixed polymers with melanins from opioid peptides.

The oxidation of oxytocin and vasopressin by peroxidase/H2O 2 system.

Oxytocin and vasopressin are oxidized by horseradish peroxidase and by lactoperoxidase, in the presence of hydrogen peroxide. Spectrophotometric measurements are indicative of the formation of dityrosine. Kinetic parameters indicate that the affinity of horseradish peroxidase is slightly higher for oxytocin with respect to vasopressin and that the two hormones are better substrates for both peroxidases than free tyrosine.

Lipoxygenase-catalyzed oxidation of catecholamines.

Dopa and structurally related catecholamines in presence of hydrogen peroxide are oxidized in vitro by soybean lipoxygenase producing the corresponding melanin pigments. The kinetic parameters of the catecholasic reaction, measured as aminochrome formation, have been calculated. The rate of peroxidation depends on catecholamine and hydrogen peroxide concentration. The optimum pH for the peroxidative activity of the enzyme is around 8.5. The enzyme, at higher pH values (pH 9-9.5), is also able to perform an oxidative reaction of the substrates. Implications of the possible biochemical relevance of the reactions are discussed.

On the oxidation of cystathionamine and selenocystathionamine by plant amineoxidase.

Cystathionamine and selenocystathionamine, diamines analogous to 1,6-diaminohexane but having the third methylene group of the carbon chain substituted by a S or a Se atom, are asymmetrical thio- (seleno-) ethers. They can give rise by oxidative monodeamination to two different aminoaldehydes. It has been shown that lentil seedlings amineoxidase catalyzes the oxidative deamination of either the one or the other aminogroup of cystathionamine or of selenocystathionamine, giving rise to both possible aminoaldehydes.

Some biochemical properties of melanins from opioid peptides.

Opioid peptides are converted by mushroom tyrosinase into melanin-like compounds retaining the peptide moiety (opio-melanins). Opio-melanins, owing to the presence of the linked aminoacids and in contrast with DOPA-melanin, are soluble compounds. The enkephalin-generated melanins are cleaved by carboxypeptidase A and pronase whereas aminopeptidase M cannot remove aminoacids from the pigment. Enkephalins, as well as other opioid peptides, (alpha-endorphin, kyotorphin, esorphins) if oxidized in presence of DOPA and tyrosinase are readily incorporated into DOPA-melanin. The resulting mixed-melanins (opio-melanin + DOPA-melanin) can be solubilized in hydrophilic solvents. Melanin from leu-enkephalin exhibits paramagnetism as evidenced by an EPR spectrum identical to that of DOPA-melanin, but unlike the latter pigment, it does not appear to oxidize NADH, probably for the presence of the peptide moiety that exerts a hampering effect on the oxidizing capacity.

Selenomethionine as substrate for glutamine transaminase.

Selenomethionine is as a good substrate as methionine for bovine liver glutamine transaminase (E.C. 2.6.1.15). Almost identical Km values for methionine, selenomethionine, 4-methylthio-2-oxobutanoic acid and 4-methylseleno-2-oxobutanoic acid have been obtained. Like for other enzymes, also for glutamine transaminase the substitution of the sulfur atom in a substrate molecule by a selenium one does not appreciably affect the enzyme affinity. Glutamine transaminase may thus be involved in selenomethionine catabolism.

The oxidation of sulfur containing cyclic ketimines The sulfoxide is the main product of S-aminoethyl-cysteine ketimine autoxidation.

The products of autoxidation of S-aminoethyl-L-cysteine ketimine (AECK) have been analysed with the amino acid analyzer, with thin layer chromatography and with high performance liquid chromatography. Under the conditions of the assay (pH 8.5, 38°C, O2 bubbling) AECK is almost totally oxidized in 1.5 hours. Among the final products a component running fast in HPLC, named Cx1, has been isolated, reduced with NaBH4 and analysed. Reduced Cx1 resulted to show the same properties of synthetic thiomorpholine-3-carboxylic acid-S-oxide, known in the past literature with the name of "chondrine". On the basis of these results and by specific chromatographic tests, Cx1 has been identified as the sulfoxide of AECK. Among the other autoxidation products, thiomorpholine-3-one has been identified. The detection, after HCl hydrolysis, of glyoxylic acid and mesoxalic semialdehyde together with cysteamine indicates that compounds provided with easily cleavable S-C bonds, possibly thiohemiacetals or (and) thioesters, are the likely intermediates for other products. AECK sulfoxide and thiomorpholine-3-one are relatively stable and cannot be taken as the main intermediates for the remaining oxidation products.

Transamination of some sulphur- or selenium-containing amino acids by bovine liver glutamine transaminase.

S-(3-aminopropyl)cysteine and Se-(3-aminopropyl)selenocysteine are deaminated by bovine liver glutamine transaminase. The corresponding alpha-keto acids, S-(3-aminopropyl)-thiopyruvic acid and Se-(3-aminopropyl)selenopyruvic acid, are produced which spontaneously cyclize to ketimine derivatives. They have been identified by comparing their UV absorption spectra and some chemical or chromatographic properties with chemically synthesized authentic samples. Also S-(2-aminoethyl)homocysteine is the substrate for the enzyme. Kinetic parameters determined in comparison to thialysine and selenalysine show that neither the presence of a sulphur or a selenium atom nor the relative position of the atom in the carbon chain appreciably affects the substrate specificity of the enzyme. However, the length of the carbon chain has some influence on it.

The peroxidase-catalyzed oxidation of enkephalins.

In vitro experiments are reported showing that Leu-enkephalin and Metenkephalin, in the presence of hydrogen peroxide, can be oxidized by horseradish peroxidase. The products formed are strongly fluorescent and characterized by absorption peaks with maxima at 290 nm and 315 nm. The effects of substrate and enzyme concentrations on the oxidation rate of enkephalins are described. Amino acid analysis of the hydrolysates from peroxidase-treated enkephalins provides evidence for the presence of dityrosine. The data suggest that the oxidation leads to the production of enkephalin dimers with a linkage between the N-terminal tyrosine residues. Data are also obtained indicating that enkephalins function as hydrogen donors for mammalian peroxidases.

The peroxidase-catalyzed oxidation of kyotorphins.

In vitro experiments are reported showing that the dipeptides Tyr-L-Arg (kyotorphin) and Tyr-D-Arg (D-Arg-kyotorphin) can be oxidized by H2O2-horseradish peroxidase system: the products formed are characterized by absorption spectra with two peaks at 290 nm and 315 nm. The effects of substrate and enzyme concentration on the oxidation rate are described. Amino acid analysis of hydrolysates of peroxidase-treated kyotorphins provides evidence for the presence of dityrosine. The data suggest that the oxidation leads to the production of dimers with an o,o-linkage between the tyrosine residues.

Thialysine utilization for protein synthesis by an exponentially growing E. coli culture.

The extent of protein lysine substitution by thialysine in E. coli cells grown in media containing the analog depends on the time interval the cells are grown in the presence of analog and on the analog concentration in the medium. By calculating the percent of lysine substitution in newly synthesized proteins it was shown that this reaches, after one cell doubling in the presence of analog, a maximum which is 17% in the cells grown with 0.1 or 0.2 mM thialysine and 8% in cells grown with 0.05 mM thialysine. Proteins synthesized in the presence of analog in the concentration range 0.05-0.2 mM show similar stability to those synthesized in the absence of analog. The extent of analog incorporation into newly synthesized proteins, as regards both the time course and the dependence on analog concentration in the medium, is strictly related to the extent of the repression of AK III, the first enzyme of lysine biosynthetic pathway.

Selenalysine transamination by a bovine brain enzyme.

Selenalysine is deaminated by glutamine transaminase from bovine brain, leading to the production of the corresponding alpha-ketoacid, which spontaneously cyclizes to a ketimine form. Selenalysine shows a good affinity for the enzyme.

Degradation of thialysine- or selenalysine-containing abnormal proteins in E. coli.

Thialysine and selenalysine can be utilized for protein synthesis by lysine-requiring E. coli cells even in the absence of lysine. Protein synthesis has been determined as labeled leucine incorporation into acid-insoluble material, as increase of cell proteins and as protein-lysine substitution by the analog. Either analog can be incorporated into proteins, in the absence of lysine, for a limited time interval after which cells stop to duplicate. Proteins synthesized during this period contain most of their lysine residues substituted by the analog. Moreover, it has been shown that the analog-containing proteins are unstable and rapidly degraded. Their instability would account for the inability of lysine-requiring E. coli cells to utilize the analog as growth factor.