Enrico Sanjust

Quantitative evaluation of oxidative stress, chronic inflammatory indices and leptin in cancer patients: correlation with stage and performance status.

In advanced cancer patients, the oxidative stress could take place either at the onset of disease or as a function of disease progression. To test this hypothesis, the following parameters were investigated: the erythrocyte activity of the enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), the serum activity of glutathione reductase (GR) and the serum total antioxidant status (TAS). The total antioxidant capacity of plasma LMWA was evaluated by the cyclic voltammetry methodology. We further determined the serum levels of proinflammatory cytokines (IL‐6 and TNFα), IL‐2, leptin and C‐reactive protein (CRP). All of these parameters have been correlated with the most important clinical indices of patients such as Stage of disease, ECOG PS and clinical response. Eighty‐two advanced stage cancer patients and 36 healthy individuals used as controls were included in the study. Our findings show that SOD activity was significantly higher in cancer patients than in controls and GPx activity was significantly lower in cancer patients than in controls. Serum values of IL‐6, TNFα and CRP were significantly higher in patients than in controls. Serum leptin values of cancer patients were significantly lower than controls. SOD activity increased significantly from Stage II/ECOG 0‐1 to Stage IV/ECOG 0–1, whereas it decreased significantly in Stage IV/ECOG 3. GPx activity decreased significantly in Stage IV/ECOG 2–3. An inverse correlation between ECOG PS and serum leptin levels was found. Serum levels of IL‐2 decreased from Stage II/ECOG 0–1 to Stage IV/ECOG 2–3. A direct correlation between Stage/ECOG PS and serum levels of both IL‐6 and CRP was observed. Cisplatin administration induced a significant increase of GPx after 24 hr. In conclusion, this is the first study that shows that several “biological” parameters of cancer patients such as antioxidant enzyme activity, cytokines, leptin and CRP strictly correlate with the most important clinical parameters of disease such as Stage and ECOG PS.

Tyrosinase inhibition: general and applied aspects.

The active site of tyrosinase is described with a view to depicting its interactions with substrates and inhibitors. Occurrence and mechanism(s) of tyrosinase-mediated browning of agrofood products are reviewed, with regard to both enzymic and chemical reactions, and their control, modulation, and inhibition. Technical and applicational implications are discussed.

Inorganic Materials as Supports for Covalent Enzyme Immobilization: Methods and Mechanisms

Several inorganic materials are potentially suitable for enzymatic covalent immobilization, by means of several different techniques. Such materials must meet stringent criteria to be suitable as solid matrices: complete insolubility in water, reasonable mechanical strength and chemical resistance under the operational conditions, the capability to form manageable particles with high surface area, reactivity towards derivatizing/functionalizing agents. Non-specific protein adsorption should be always considered when planning covalent immobilization on inorganic solids. A huge mass of experimental work has shown that silica, silicates, borosilicates and aluminosilicates, alumina, titania, and other oxides, are the materials of choice when attempting enzyme immobilizations on inorganic supports. More recently, some forms of elemental carbon, silicon, and certain metals have been also proposed for certain applications. With regard to the derivatization/functionalization techniques, the use of organosilanes through silanization is undoubtedly the most studied and the most applied, although inorganic bridge formation and acylation with selected acyl halides have been deeply studied. In the present article, the most common inorganic supports for covalent immobilization of the enzymes are reviewed, with particular focus on their advantages and disadvantages in terms of enzyme loadings, operational stability, undesired adsorption, and costs. Mechanisms and methods for covalent immobilization are also discussed, focusing on the most widespread activating approaches (such as glutaraldehyde, cyanogen bromide, divinylsulfone, carbodiimides, carbonyldiimidazole, sulfonyl chlorides, chlorocarbonates, N-hydroxysuccinimides).

Complete and efficient enzymic hydrolysis of pretreated wheat straw

Abstract A fractionation of wheat straw components in a two-step chemical pretreatment is proposed. Hemicelluloses were hydrolysed by dilute sulphuric acid, allowing a substantial recovery of crystalline xylose. Lignin was removed by means of a mild alkaline/oxidative solubilisation procedure, involving no sulphite or chlorine and its derivatives. The use of diluted reagents and relatively low temperatures, was both cheap and environmentally friendly. The pretreated material was nearly pure cellulose, whose enzymic hydrolysis proceeded fast and with high yields, leading to high glucose syrups of remarkable purity.

Mild alkaline/oxidative pretreatment of wheat straw

A new mild alkaline/oxidative pretreatment of wheat straw prior to enzymic hydrolysis was carried out. It consists of a first alkaline (1% NaOH for 24 h) step, which mainly solubilises hemicellullose and renders the material more accessible to further chemical attack, and a second alkaline/oxidative step (1% NaOH and 0·3% H2O2 for 24 h), which solubilises and oxidises lignin to minor polluting compounds. The entire process was carried out at low temperature (25–40°C) using a low concentration of chemicals, resulting in a relatively low cost and waste liquors containing only trace amounts of dangerous pollutants derived from lignin. Recovery of cellulose after the double pretreatment reached 90% of that contained in the starting material, with a concomitant 81% degradation of lignin. The action of a commercial cellulase on the cellulose obtained produced a syrup with a high concentration of reducing sugars (220 mg/ml), of which a large percentage was glucose.

Olive milling wastewater as a medium for growth of four Pleurotus species.

Four species ofPleurotus were adapted to grow on olive milling wastewater, and in certain conditions produced high yield of fruit bodies. Some biochemical transformations were observed in the olive milling wastewater owing to the growth ofPleurotus. In particular, the fungi actively excreted large amounts of laccase in the medium, and at the same time the concentration of phenolics and other toxic compounds significantly decreased, as revealed by HPLC analysis and toxicity tests on standard cultures of human cell lines.

Agarose and Its Derivatives as Supports for Enzyme Immobilization

Agarose is a polysaccharide obtained from some seaweeds, with a quite particular structure that allows spontaneous gelation. Agarose-based beads are highly porous, mechanically resistant, chemically and physically inert, and sharply hydrophilic. These features—that could be further improved by means of covalent cross-linking—render them particularly suitable for enzyme immobilization with a wide range of derivatization methods taking advantage of chemical modification of a fraction of the polymer hydroxyls. The main properties of the polymer are described here, followed by a review of cross-linking and derivatization methods. Some recent, innovative procedures to optimize the catalytic activity and operational stability of the obtained preparations are also described, together with multi-enzyme immobilized systems and the main guidelines to exploit their performances.

Effects of plant-derived naphthoquinones on the growth of Pleurotus sajor-caju and degradation of the compounds by fungal cultures

The growth of the white‐rot basidiomycete Pleurotus sajor‐caju in malt‐agar plates was inhibited by three naturally occurring, plant‐derived naphthoquinones: juglone, lawsone, and plumbagin. The latter two compounds exerted the most potent antifungal activity, and lawsone killed the mycelium at concentrations higher than 200 ppm. Plates containing juglone and lawsone presented large decolorized areas extending from area of fungal growth, suggesting an extracellular enzymatic degradation of these quinones. Screening of culture plates for extracellular enzymatic activities revealed the presence of both laccase and veratryl alcohol oxidase in most plates, the diffusion of both enzymes matching the decolorized area. In agitated cultures, the presence of juglone was found to stimulate the production of veratryl alcohol oxidase in a significant manner. This is the first time degradation of plant derived naphthoquinones by a white‐rot fungus is reported.

Effect of 3-hydroxyanthranilic acid on mushroom tyrosinase activity

Tyrosinase is a copper containing protein which catalyzes the hydroxylation of monophenols and the oxidation of diphenols to o-quinones. The monophenolase activity of tyrosinase is characterized by a typical lag time. In this paper the influence of 3-hydroxyanthranilic acid on monophenolase activity of tyrosinase is reported. 3-Hydroxyanthranilic acid reduced the lag time of tyrosinase when the enzyme acted on N-acetyl-L-tyrosine and on 4-tert-butylphenol. In the presence of 3-hydroxyanthranilic acid, the reaction product 4-tert-butyl-o-benzoquinone, derived from 4-tert-butylphenol oxidation, was formed at a higher rate than in its absence. The results reported in this paper indicate that 3-hydroxyanthranilic acid could affect the enzymic activity of mushroom tyrosinase probably by acting as a diphenol substrate. A K(m) value of 0.78 mM was calculated for 3-hydroxyanthranilic acid as substrate. When tyrosinase acted on 4-tert-butylphenol, K(m) for 3-hydroxyanthranilic acid as a cofactor was estimated to be 37.5 microM. No effect was observed on the diphenolase activity of the enzyme acting on 4-tert-butylcatechol in the presence of 3-hydroxyanthranilic acid.

Detection of Laccase, Peroxidase, and Polyphenol Oxidase on a Single Polyacrylamide Gel Electrophoresis

Abstract Laccase, polyphenol oxidase and peroxidase are widely distributed in both plants and micro-organisms. Among these enzymes, the identification of one particular activity in the presence of the others is often difficult as enzymes can often oxidise the same substrates. A method is described, that is suitable to differentiate the three activities on the same polyacrylamide gel electrophoresis. The method is based on the use of two substrates, 4-aminoN,N-diethylaniline and 4-tert-butyl-catechol, so that coloured spots appear corresponding to the different activities. Moreover, a comparison among different mushroom species belonging to the same genus is reported using the described method.