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Ocean Acidification (OA) is likely to affect macroalgal diversity in the future with species-specific responses shaping macroalgal communities. In this framework, it is important to focus research on the photosynthetic response of habitat-forming species which have an important structural and functional role in coastal ecosystems. Most of the studies on the impacts of OA involve short-term laboratory or micro/mesocosm experiments. It is more challenging to assess the adaptive responses of macroalgal community to decreasing ocean pH over long-term periods, as they represent the basis of trophic dynamics in marine environments. This work aims to study the physiological traits of a population of Sargassum vulgare that lives naturally in the high pCO 2 vents system in Ischia (Italy), in order to predict the species behaviour in a possible OA future scenario. With this purpose, the photosynthetic performance of S. vulgare was studied in a wild, natural population living at low pH (6.7) as well as in a population transplanted from native (6.7) to ambient pH (8.1) for three weeks. The main results show that the photochemical activity and Rubisco expression decreased by 30% after transplanting, whereas the non-photochemical dissipation mechanisms and the photosynthetic pigment content increased by 50% and 40% respectively, in order to compensate for the decrease in photochemical efficiency at low pH. Our data indicated a stress condition for the S. vulgare population induced by pH variation, and therefore a reduced acclimation capability at different pH conditions. The decline of the PS II maximum quantum yield (F v /F m ) and the increase of PARP enzyme activity in transplanted thalli further supported this hypothesis. The absence of the species at ambient pH conditions close to the vent system, as well as the differences in physiological traits, suggest a local adaptation of S. vulgare at pH 6.7, through optimization of photosynthetic performance.

Background Reactive oxygen species (ROS) in biological tissues of elected biosentinels represent an optimal biomarker for eco-monitoring of polluted areas. Electron spin resonance (ESR) is the most definitive method for detecting, quantifying and possibly identifying radicals in complex systems. Objective A non-invasive method for monitoring polluted areas by the quantitative determination of ROS in frog skin biopsy is presented. Methods We assessed by ESR spectroscopy the ROS level in adult male of Pelophylax bergeri, specie not a risk of extinction, collected from the polluted Sarno River (SA, Italy) basin. The spin-trap ESR method was validated by immunohistochemical analysis of the well-assessed pollution biomarkers cytochrome P450 aromatase 1A (CYP1A) and glutathione S-transferase (GST), and by determining the poly(ADPribose) polymerase (PARP) and GST enzymatic activity. Results ROS concentration in skin samples from frogs collected in the polluted area is significantly higher than that determined for the unpolluted reference area. Immunohistochemical analysis of CYP1A and GST supported the reliability of our approach, even in the absence of evident morphological and ultrastructural differences. PARP activity assay, connected to possible oxidative DNA damage, and the detoxification index by GST enzymatic assay give statistically significant evidence that higher levels of ROS are associated to alterations of the different biomarkers. Conclusions ROS concentration, measured by ESR on isolated frog skin, through the presented non-lethal method, is a reliable biomarker for toxicity screening and represents a useful basic datum for future modelling studies on environmental monitoring and biodiversity loss prevention.

In a growing deterioration of the ecosystem both in terms of biodiversity and ecological features, even more problematic is the conservation of species. The aim of this work was to study seasonal variations of biochemical and molecular DNA damage markers in Pelophylax bergeri exposed to potential endocrine disrupting chemicals. Frogs were collected in the main phase of the reproductive cycle: in April and May, during active spermatogenesis and the breeding season, and in October and November, in spermatogenetic regression, from a sampling site in the polluted Sarno River and from a clean reference site (Matese Lake). DNA profile and poly(ADPribose) polymerase (PARP) activity were evaluated in the testis. In the main phases of the reproductive cycle, all specimens from Sarno River showed a PARP activity higher than that measured in the same frog’s testis from Matese Lake. In addition, the PARP activity in active spermatogenesis was always higher compared with the activity in the spermatogenetic regression in Sarno River frogs. PARP2, usually activated in response to exposure to heavy metals, was expressed in all testes. In the species examined from Sarno River, no evident correlation between testis DNA damage and PARP activation was found. The working hypothesis is that PARP, implicated in genome surveillance and protection, might represent in frog spermatogenesis an appealing tool for genotoxic risk assessment useful to define a warning alarm for its survival.

The behaviour of three different laccase-based graphite biosensors was studied in view of their use in agricultural or industrial waters polluted by phenolic compounds. Catechol was used as a substrate model. Laccase from Trametes versicolor was immobilized on one biosensor (type A electrode) by adsorption while, on the other two biosensor types, laccase was covalently bound through the carboxylic groups created on the graphite by means of treatment with an electric potential difference (type B electrode) or with nitric acid (type C electrode). In the latter two cases, hexamethylenediamine and glutaraldehyde were used as the spacer and the coupling agent, respectively. The extension of linear response range and the sensitivity and time stability of each biosensor type were investigated. The type C biosensor gave the best results and its electrochemical properties proved comparable to those reported by other authors.

Poly(ADPribosyl)ation, catalysed by poly(ADP-ribose)polymerases, affects many cellular events and has a recognized epigenetic role. Nuclear poly(ADP-ribose)polymerases 1 and 2 are hyper activated by DNA strandbreaks. They auto-modify with large polymers of ADP-ribose and recruit DNA repair proteins. The more the DNA strand-breaks, the more poly(ADP-ribose) polymerase modifies itself. On the other hand, dietary lipids can be signaling molecules, lead to pro-(ω6)/ anti-(ω3) inflammatory compounds, and be included in biomembranes, good biomarkers of their unbalance. Here, we report the results obtained from an epidemiological study to establish whether the combination of two different analyses, i.e. detecting auto-modified poly (ADP-ribose) polymerase levels and analyzing erythrocyte membrane fatty acid composition, might help to monitor the physio-pathological state of the cell, and to correlate with lifestyle, diet or diseases. The two analyses were carried blindly on 70 subjects undergoing endoscopy. They were first interviewed, to collect anamnesis and clinical data, if present. Lymphocytes and erythrocytes were prepared from venous blood to assay poly (ADP-ribose) polymerase auto modification and membrane fatty acid content, respectively. The results were statistically evaluated. The measure of poly (ADP-ribose) polymerase automodification confirmed that its levels correlate with DNA damage extent, within the same pathology, and allowed to monitor the clinical activity of the disease, depending on ongoing therapeutic/surgical treatment. Membrane fat profile was able to evidence unbalance of lipids linked to both diet/lifestyle and inflammatory states leading to diseases. Both analyses provide possible biomarkers for sensible, non-invasive and routine monitoring.

A hydrophobic and catalytic membrane has been obtained by grafting with γ -radiations a Teflon membrane in presence of methylmethacrylate solution and by immobilizing on it, after treatment with glutaraldehyde, the β-Galactosidase enzyme. When employed in a non-isothermal bioreactor, the membrane showed an increase in catalytic activity proportional to the applied temperature gradients. The results have been explained in terms of distinct contributions from the process of thermodialysis and the conformational changes induced in the dynamic structure of the enzyme by the presence of heat flow. The increase of the yield of the process has been evaluated in terms of a coefficient α representing the percent increase of enzyme activity when a temperature difference of 1°C is applied across the membrane. The catalytic Teflon membrane used here gave values of 20%, which are comparable to those obtained with other membrane systems, making this kind of membrane useful for practical applications in industrial processes.

The scenarios for the long-term habitation of space platforms and planetary stations involve plants as fundamental part of Bioregenerative Life Support Systems (BLSS) to support the crew needs. Several constraints may limit plant growth in space: among them ionizing radiation is recognized to severely affect plant cell at morphological, physiological and biochemical level. In this work, plants of Phaseolus vulgaris L. were subjected to four different doses of X-rays (0.3, 10, 50 and 100 Gy) in order to assess the effects of ionizing radiation on this species and to analyze possible mechanisms carried out to overcome the radiation injuries. The effects of X-rays on plant growth were assessed by measuring stem elongation, number of internodes and leaf dry weight. The integrity of photosynthetic apparatus was evaluated by photosynthetic pigment composition and ribulose 1,5-bisphosphate carboxylase (Rubisco) activity, whereas changes in total antioxidant pool and glutathione S transferase activity (GST) were utilized as markers of oxidative stress. The distribution of phenolic compounds in leaf tissues as natural shielding against radiation was also determined. Irradiation of plants at 0.3 and 10 Gy did not determine differences in all considered parameters as compared to control. On the contrary, at 50 and 100 Gy a reduction of plant growth and a decrease in photosynthetic pigment content, as well as an increase in phenolic compounds and a decrease in total antioxidant content and GST activity were found. Only a slight reduction of Rubisco activity in leaves irradiated at 50 and 100 Gy was found. The overall results indicate P. vulgaris as a species with a good potential to face ionizing radiation and suggest its suitability for utilization in BLSSs.

The controversy about the occurrence of an (ADPribosyl)ating activity in yeast is still standing up. Here we discuss this topic on the basis of results obtained with classic experiments proposed over years as basis to characterize an (ADPribosyl)ation system in any organism. Independent results obtained in two different laboratories were in line with each other and went towards the occurrence of an active (ADPribosyl)ating system in Saccharomyces cerevisiae. In fact data collected from nuclear preparations of cultured cells matched those from baker's yeast and lyophilized yeast cells. Yeast (ADPribosyl)ating enzyme is a protein of 80–90 kDa, as determined by electrophoresis on polyacrylamide gel in sodium dodecyl sulphate, followed by immunoblotting with antibodies against anti-poly(ADPribose) polymerase catalytic site. It synthesizes products, that, after digestion with phosphodiesterase, co-migrates mainly with phosphoribosyl adenosine monophosphate after thin layer chromatography on silica gel plate. © 2005 Wiley-Liss, Inc.

In view of the treatment of agricultural waste waters, urea polluted, a catalytic and hydrophobic membrane was constructed by immobilizing urease on a nylon sheet grafted with cyclohexyl methacrylate (CHMA). Hexamethylenediamine (HMDA) and glutaraldehyde (GA) were used as spacer and crosslinking agent, respectively. With reference to the soluble counterpart immobilized urease was found to exhibit (i) a shift of the optimum pH towards more acidic values; (ii) a shift of the optimum temperature towards higher temperatures; (iii) higher values of Km. The latter result indicated an apparent loss of affinity of immobilized urease towards urea. To recovery this affinity loss, the catalytic membranes were employed in a bioreactor operating under non-isothermal conditions. Under these conditions the catalytic membranes exhibited reaction rates higher and apparent Km smaller than those measured under comparable isothermal conditions. As a consequence, percentage increases of enzyme activity and reduction of the production times, proportional to the magnitude of the applied temperature difference were observed. Results have been discussed in the frame of reference of the process of thermodialysis. The technology of the non-isothermal bioreactors confirmed its usefulness also in the reduction of urea concentration in aqueous solutions.

Glucose oxidase (GOD) was immobilized on nylon membranes having three different pore diameters and chemically grafted with glycidyl methacrylate (GMA) or butyl methacrylate (BMA). Hexamethylenediamine (HMDA) and glutaraldehyde (GA) were used as spacer and coupling agent, respectively. The biochemical and electrochemical behaviour of the membranes has been studied as a function of pH, temperature and glucose concentration with reference to the grafted monomer and the membrane pore diameter. The behaviour of the soluble GOD has also been studied in order to see the modification induced by the immobilization process on the enzyme activity. It was found that the values of the biosensor sensitivity, maximum saturation current and electrochemical affinity increase with the membrane pore diameter, indipendently of the nature of the graft monomer. Opposite behaviour was found relatively to the extension of the linear response ranges and the average response times. With reference to the parameters increasing with the pore diameter it was found that membranes grafted with GMA had higher values than those of the membranes grafted with BMA. The contrary occurred to the values of the parameters decreasing with the increase of the pore diameter. Biochemical and electrochemical results have been discussed in terms of the different limitations to the diffusion of substrate and reaction products across the catalytic membrane introduced by the different pore diameters and by the different hydrophobicity of the graft monomers.

The behavior of three different catalytic membranes, obtained by immobilizing urease on nylon sheets chemically grafted with methyl methacrylate, was studied in a bioreactor operating under isothermal and non-isothermal conditions. Membrane activation was carried out by condensation or acyl azide reaction, and spacers of different lengths, such as hexamethylendiamine or hydrazine, were used. Under isothermal conditions, the activities of the catalytic membranes and soluble urease were characterized as a function of pH, temperature, and urea concentration. Both enzyme forms showed the same optimum pH, whereas the optimum temperature was lower for the immobilized enzymes. The spacer length appeared to determine broader pH- and temperature-activity profiles for the urease derivatives. The apparent K(m) values of the insoluble urease were dependent on membrane type and were higher than those of the soluble counterpart, thus indicating an affinity loss for urea. Under non-isothermal conditions, all membranes exhibited an increase of percentage activity proportional to the applied temperature difference and decreasing with the increase of urea concentrations. A decrease of the apparent K(m) was also observed. These results suggest that substrate diffusion limitations due to the immobilization process can be overcome in the presence of temperature gradients. In addition, the remarkable reduction of the production times supports the use of non-isothermal bioreactors for the treatment of urea-polluted waste waters.

Poly(ADP-ribosylation) is a post-translational modification of proteins involved in a wide range of molecular and cellular processes in mammalian system. The main enzymes responsible for this modification are the poly(ADP-ribose) polymerases that catalyze the transfer of ADP-ribose moieties from NAD + to target protein acceptors, producing long and branched ADP-ribose polymers. The poly(ADP-ribosyl)ation is rapidly reverted by poly(ADP-ribose) glycohydrolase enzymes, which hydrolyzes poly(ADP-ribose) polymers, generating free ADP-ribose. So far, nine proteins with a poly(ADP-ribose) polymerase signature and two poly(ADP-ribose) glycohydrolase enzymes encoded by two adiacent genes were identified in Arabidopsis . The present review will describe the structures and functions of plant poly(ADP-ribose) polymerases and poly(ADP-ribose) glycohydrolases.

β-Galactosidase was immobilized on a nylon membrane chemically grafted with butylmethacrylate. Hexamethylenediamine was used as spacer and glutaraldehyde as coupling agent. The catalytic behaviour of the membrane was studied under isothermal and non-isothermal conditions. It was found that in the presence of temperature gradients, the catalytic membrane exhibited higher activity and greater affinity for the substrate. The increases of the enzyme reaction rate was found to be proportional to the intensity of the applied temperature gradient. Percentage increases of the activity were also found to decrease with the increase of the average temperature and concentration. All the results are phenomenologically discussed in terms of substrate traffic across the catalytic membrane induced by the processes of diffusion or thermodialysis under isothermal and non-isothermal conditions, respectively.

A new hydrophobic and catalytic membrane was prepared by immobilizing β-galactosidase on a polytetrafluoroethylene (PTFE) membrane, double grafted by means of γ-radiation. Methacrylic acid (MAA) and 2-hydroxyethyl methacrylate (HEMA) were used as monomers to be grafted. 1-6 Hexamethylendiamine (HMDA) and glutaraldehyde (GA) were used as spacer and coupling agent, respectively. The membranes were characterized physically and biochemically. The values of the hydraulic and thermoosmotic permeability coefficients were calculated. The dependence of catalytic membrane activity on the temperature, pH and substrate concentration was studied. Lactose hydrolysis, performed under isothermal and non-isothermal conditions, confirmed the usefulness of employing non-isothermal bioreactors in industrial processes. In particular, characteristic parameters of the non-isothermal process, such as α and β, indicative of the efficiency increase of the yield of an enzymatic reaction when a difference of temperature of 1°C is applied across the membrane, were calculated. These values were compared with those obtained so far with other membrane systems.

In the archaeon Sulfolobus solfataricus, protein ADPribosylation by free ADPribose was demonstrated by testing both [adenine-14C(U)]ADPR and [adenine- 14C(U)]NAD as substrates. The occurrence of this process was shown by using specific experimental conditions. Increasing the incubation time and lowering the pH of the reaction mixture enhanced the protein glycation by free ADPribose. At pH 7.5 and 10 min incubation, the incorporation of free ADPribose into proteins was highly reduced. Under these conditions, the autoradiographic pattern showed that, among the targets of ADPribose electrophoresed after incubation with 32P-NAD, the proteins modified by free 32P-ADPribose mostly corresponded to high molecular mass components. Among the compounds known to inhibit the eukaryotic poly-ADPribose polymerase, only ZnCl2 highly reduced the ADPribose incorporation from NAD into the ammonium sulphate precipitate. A 20% inhibition was measured in the presence of nicotinamide or 3-aminobenzamide. No inhibition was observed replacing NAD with ADPR as substrate. J. Cell. Biochem. 66: 37–42, 1997. © 1997 Wiley-Liss, Inc.

Ten different strains of unicellular microalgae have been used in the bioconversion of progesterone. Regio and stereoselective reduction and hydroxylation of the title compound were induced. A 9,10-seco derivative was obtained in high yield by using Scenedesmus quadricauda. © 1994.