Anna De Maio

NEMO-binding domain peptide inhibits proliferation of human melanoma cells

Melanoma is the most aggressive form of skin cancer, it originates from melanocytes and its incidence has increased in the last decade. Recent advances in the understanding of the underlying biology of the progression of melanoma have identified key signalling pathways that are important in promoting melanoma tumourigenesis, thus providing dynamic targets for therapy. One such important target identified in melanoma tumour progression is the Nuclear Factor-kappaB (NF-kappaB) pathway. In vitro studies have shown that NF-kappaB binding is constitutively elevated in human melanoma cultures compared to normal melanocytes. It has been found that a short cell-permeable peptide spanning the IKK-beta NBD, named NBD peptide, disrupted the association of NEMO with IKKs in vitro and blocked TNFalpha-induced NF-kappaB activation in vivo. In the present study we investigated the effect of the NBD peptide on NF-kappaB activity and survival of A375 human melanoma cells. We found that NBD peptide is able to inhibit the proliferation of A375 cells, which present constitutively elevated NF-kappaB levels. Inhibition of cell proliferation by NBD peptide was associated with direct inhibition of constitutive NF-kappaB DNA-binding activity and induction of apoptosis by activation of caspase-3 as confirmed by the cleavage and consequently inactivation of poly (ADP ribose) polymerase (PARP-1) known as the best marker of this process.

The ADP-ribosylating thermozyme from Sulfolobus solfataricus is a DING protein

Abstract The partial amino acid sequence of the sulfolobal thermoprotein biochemically characterized as poly(ADP-ribose)polymerase-like enzyme overlaps those of DING proteins. This group of proteins, widely occurring in animals, plants and eubacteria, shows a characteristic and highly conserved N-terminus, DINGGGATL. The sequence of the N-terminal region and of the analyzed tryptic peptides of the sulfolobal thermozyme shows a high similarity with most of the DING proteins from databases. This is the first example of a DING protein from a sulfolobal source.

ADP-ribosylation reactions in Sulfolobus solfataricus, a thermoacidophilic archaeon

An ADP-ribosylating system was detected in a crude homogenate from Sulfolobus solfataricus, a thermophilic archaeon, optimally growing at 87 degrees C. The archaeal ADP-ribosylation reaction was time-, temperature- and NAD-dependent. It proved to be highly thermostable, with about 30% decrease of 14C incorporation from [14C]NAD on incubation at 80 degrees C for up to 24 h. The main reaction product was found to be mono-ADP-ribose. Testing both [adenine-14C(U)]NAD and [adenine-14C(U)]ADPR as substrates, it was found that acceptor proteins were modified by ADP-ribose both enzymatically, via ADP-ribosylating enzymes, and via chemical attachment of free ADP-ribose, likely produced by NAD glycohydrolase activity. The synthesis of ADP-ribose-protein complexes was shown to involve mainly acceptors with molecular masses in the 40-100 kDa range, as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate.

Assessment of Eco-Physiological Performance of Quercus ilex L. Leaves in Urban Area by an Integrated Approach

Plant physiological performance may be endangered in contaminated urban areas. Metal and polycyclic aromatic hydrocarbon (PAH) content in Quercus ilex leaves mainly reflects air pollution, being for these pollutants traceable the translocation from soil. In this work, in a field study, the responses at structural and functional level of Q. ilex leaves to metal and PAH pollution were assessed by an integrated approach. At this purpose, morphological (functional traits), biochemical (pigment content, antioxidant capacity, and PARP activity), and physiological (partitioning of absorbed light in the photosynthetic process) analyses were performed in leaves collected at urban parks and roadsides. Compared to the leaves of the urban parks, those sampled at the roadsides showed higher metal and PAH accumulation. The most contaminated leaves showed smaller leaf sizes and lower relative water and pigment content, as well as a lower photochemistry. In this circumstance, the excess of absorbed light at PSII was mainly dissipated via non-regulated than heat processes. On the basis of the biochemical analyses, the occurrence of DNA damages in contaminated Q. ilex leaves may be hypothesized.

Characterization of Novel Cytoplasmic PARP in the Brain of Octopus vulgaris

Recent investigation has focused on the participation of the poly (ADP-ribose) polymerase (PARP) reaction in the invertebrate central nervous system (CNS) during the process of long-term memory (LTM). In this paper, we characterize, localize, and assign a possible role to a cytoplasmic PARP in the brain of Octopus vulgaris. PARP activity was assayed in optic lobes, supraesophageal mass, and optic nerves. The highest levels of enzyme were found in the cytoplasmic fraction. Hyper-activation of the enzyme was detected in Octopus brain after visual discrimination training. Finally, cytoplasmic PARP was found to inhibit Octopus vulgaris actin polymerization. We propose that the cytoplasmic PARP plays a role in vivo to induce the cytoskeletonal reorganization that occurs during learning-in

Physiological responses of a population of Sargassum vulgare (Phaeophyceae) to high pCO2/low pH: implications for its long-term distribution.

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 pCO2 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 PSII maximum quantum yield (Fv/Fm) 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 pH6.7, through optimization of photosynthetic performance.

Vault-poly-ADP-ribose polymerase in the Octopus vulgaris brain: a regulatory factor of actin polymerization dynamic.

Our previous behavioural, biochemical and immunohistochemical analyses conducted in selected regions (supra/sub oesophageal masses) of the Octopus vulgaris brain detected a cytoplasmic poly-ADP-ribose polymerase (more than 90% of total enzyme activity). The protein was identified as the vault-free form of vault-poly-ADP-ribose polymerase. The present research extends and integrates the biochemical characterization of poly-ADP-ribosylation system, namely, reaction product, i.e., poly-ADP-ribose, and acceptor proteins, in the O. vulgaris brain. Immunochemical analyses evidenced that the sole poly-ADP-ribose acceptor was the octopus cytoskeleton 50-kDa actin. It was present in both free, endogenously poly-ADP-ribosylated form (70kDa) and in complex with V-poly-ADP-ribose polymerase and poly-ADP-ribose (260kDa). The components of this complex, alkali and high salt sensitive, were purified and characterized. The kind and the length of poly-ADP-ribose corresponded to linear chains of 30-35 ADP-ribose units, in accordance with the features of the polymer synthesized by the known vault-poly-ADP-ribose polymerase. In vitro experiments showed that V-poly-ADP-ribose polymerase activity of brain cytoplasmic fraction containing endogenous actin increased upon the addition of commercial actin and was highly reduced by ATP. Anti-actin immunoblot of the mixture in the presence and absence of ATP showed that the poly-ADP-ribosylation of octopus actin is a dynamic process balanced by the ATP-dependent polymerization of the cytoskeleton protein, a fundamental mechanism for synaptic plasticity.

Purification of the Poly-ADP-Ribose Polymerase-Like Thermozyme from the Archaeon Sulfolobus solfataricus

Several different protocols have been developed to purify the ADP-ribosylating enzyme from Sulfolobus solfataricus. A number of techniques have been applied in regard to the crude homogenate preparation and protein extraction. Either mechanical cell lysis with DNAase digestion or freeze-thawing with sonication allowed to obtain fairly similar amounts of the thermozyme in the homogenate. While similar recovery of thermozyme was obtained by employing both purification protocols, the proteins were solubilized with different methods, and the affinity chromatography on NAD-Agarose of the first protocol was replaced by a gel filtration step in the second protocol. When enzyme activity was compared with electrophoresis and anti-poly-ADP-ribose polymerase 1 antibody immunoblotting results, it was noticed that lysis by sonication induces aggregation of monomeric PARP-like thermozyme at least in a dimeric form. The dimeric aggregate is also evidenced by treatment of cells with sonication followed by different protein extraction (Method III). Finally, we describe the third purification protocol that allows fast recovery of small amounts of purified ADP-ribosylating enzyme.

Yeast (ADPribosyl)ation: Revisiting a controversial question

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 bakers 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.

Electron Spin Resonance (ESR) for the study of Reactive Oxygen Species (ROS) on the isolated frog skin (Pelophylax bergeri): A non-invasive method for environmental monitoring

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. HighlightsROS levels in frog skin biopsy were determined by spin‐trapping ESR spectroscopy.Skin of frogs collected in polluted areas contains higher ROS concentration.ROS levels determined in skin biopsy follow the same trend of those from liver samples.Immunohistochemical analyses and enzymatic activity tests validate the method.ROS concentration determined by ESR in biosentinel biopsy is an effective biomarker.