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The aim of this study was to evaluate the Sn, Cd, Pb, Se, Ba, Cr, Cu, Ni, Be, Al, V, Fe, Mn, Sb concentrations in women's hair childbearing age (20¬ 30 years) along a litoral area, which falls into two provinces of Campania Region: Naples and Caserta. This area is subject to a high human impact. In addition, since the hair allows us to evaluate the different inputs linked both endogenous contaminations (deposition through the blood) and exogenus (deposition of airborne particulate matter and/or cosmetic products used), it was also possible to evaluate them. Preliminary data have shown a not uniform accumulation. An endogenous contamination for Cd, Pb, Ba, Cu, Ni, Be, Al, V, Fe, Mn, Sb; an exogenous for Cr; and a mixed one for Se and Sn were detected. Through Kruskal-Wallis (p) test significance coefficient, the data showed a clear and significant accumulation of Sn, Cd, Se, Ni, Al, V and Sb.

This paper describes the cloning of the genes coding for each component of the complex of toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization. Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined. The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA)2, endowed with hydroxylase activity. Tomo F component is an NADH oxidoreductase. The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one [2Fe-2S] cluster, and exhibits a typical flavodoxin absorption spectrum. Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence. We have shown that this depends on minor errors in the gene sequence that we have corrected. C component is a Rieske-type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one [2Fe-2S] cluster. The cluster is very sensitive to oxygen damage. Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p-cresol into 4-methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified. Finally, experimental evidence is reported which strongly support a model for the electron transfer. Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H subcomplex, and eventually to molecular oxygen

Polychlorinated biphenyls (PCBs) are toxic persistent organic pollutants present in the hydrosphere. Mussels are used as sentinel organisms for the monitoring of marine pollution for their ability to bioaccumulate contaminants, their sessile nature, filter-feeding habits and widespread distribution. Alteration in protein expression is often indicative of toxic exposure or effect and changes in protein expression profiles derived from environmental stress may be used for the identification of novel biomarkers. In this study, proteomics has been used in mussels as a preliminary screening of quantitative changes in protein expression caused by a mixture of PCBs, potentially useful as new biomarkers, following the identification of the proteins more drastically altered. Our data demonstrated that proteomic approach represents a valuable tool for identifying biomarkers of exposure to environmental contaminants.

Four new oleanane saponins (1-4), together with three known saponins, were isolated from the aerial parts of Dizygotheca elegantissima R. Vig. & Guillaumin. Their structures were elucidated by 1D and 2D NMR experiments including 1D TOCSY, DQF-COSY, HSQC, and HMBC spectroscopy, as well as ESIMS analysis. The antiproliferative activity of all isolated compounds was evaluated.

This study is mainly focused on the assembly of the DNA polymerase III catalytic core of Escherichia coli. This enzyme is responsible for the chromosome replication and it is constituted of multiple subunits. In particular, the polymerase and the exonuclease (proofreading) activities are located into two different subunits: (coded by dnaE gene) and , (coded by dnaQ). The catalytic core is composed also of the subunit with an accessory role and a dispensable nature. On the contrary, the dnaE and dnaQ are essential genes and their interaction is crucial for the assembly of a DNA polymerase III active enzyme. Several data are available on the properties of the DNA polymerase III in vitro, while little information concerns the assembly in vivo of this enzyme. The present work investigated the interaction of alpha and epsilon subunits by using different truncated forms produced in vivo, in order to identify which residues of epsilon (DnaQ) are essential for binding to alpha. Moreover, the stability of the mutant proteins was analyzed and we found that the C-terminal region of epsilon is labile and affected by proteolysis. In addition, different factors (proteases and chaperones) potentially involved in the regulation of epsilon stability have been studied.

A family of neurodegenerative diseases is associated with anomalous expansion of a polyglutamine tract in the coding region of the corresponding proteins. The current working hypothesis is that polyglutamine diseases are caused by misfolding and aggregation of the proteins with a process dictated by the polyglutamine tracts, although increasing evidence suggests an involvement of the protein context in modulating these properties. Here, we show that the AXH domain of ataxin-1, the protein involved in spinocerebellar ataxia type-1, is the region responsible for the transcriptional repression activity of ataxin-1 and participates in protein aggregation. In vitro, the isolated domain undergoes a conformational transition towards a beta-enriched structure associated with aggregation and amyloid fibre formation spontaneously and without need for destabilizing conditions. Using a transfected cell line, we demonstrate that, while determined by polyglutamine expansion, ataxin-1 aggregation is noticeably reduced by deletion of AXH or by replacement with the homologous sequence from the transcription factor HBP1, which has no known tendency to aggregate. These results provide the first direct evidence of an involvement of a region other than the polyglutamine tract in polyglutamine pathologies.

Trans-4-hydroxy-2-nonenal (HNE) is one of the major terminal products both of chemically induced and of endogenous peroxidation of n-6 unsaturated fatty acids, so that it is frequently used as an indicator of the oxidative stress 'in vivo'. HNE is considered to be not only a potent cytotoxic and genotoxic compound, as evidenced by 'in vitro' studies, but even, owing to his existence at low concentrations also in normal cells, a messenger for cell signalling in physiological conditions (1). In addition, it has been showed that the concentration of lipid peroxidation products is relatively lower in undifferentiated, highly proliferating tumor cells with respect to normal cells, and it has been hypothesized that such products may play an important role in the down-regulation of cell proliferation. The electrophilic nature of HNE explains its chemical reactivity towards thiols, aminogroups of aminoacids, proteins or DNA bases (2). Previously we showed that, after exogenous administration to a human osteosarcoma cell line, SaOS2, HNE nuclear concentration increases about threefold and that such an increase correlates with cytotoxic and cytostatic effects [3]. In this work we have developed a method to allow a good separation of the single HNE-protein adducts. The accurate quantification of such adducts provides a powerful tool for the identification of molecular targets which could mediate the aldehyde effects. The HNE-protein adducts were reduced with NaB[3H]H4 to stabilize them and allow their autoradiographic detection. The nuclear extracts were analyzed in SDS-PAGE; in the first step of analysis a 12,5 % gel and Tris-Glicine running buffer was used to obtain the separation of all proteins extracted. In the second step a 10% polyacrilamide gel was employed with MOPS and MES running buffers; each buffer produced a different migration pattern when used with the same gel. In particular we observed that to optimize the separation of proteins ranging from 21 kDa to 66 kDa it was best to use MOPS running buffer; on the other hand to resolve proteins ranging from 2.5 kDa to 36 kDa the MES running buffer was the most indicated. Finally the large molecular weight proteins (400 kDa to 55 kDa) were separated using 4-8% polyacrlamide gel with Tris Acetate SDS running buffer. After autoradiography, the bands corresponding to the protein-adducts were excised and their radioactivity was evaluated by beta-counting. The results show that the method presented here is able to separate HNE-protein adducts from distinct intracellular compartments and it could be an efficient preparatory procedure for identification of modified protein in mass spectrometry. In particular, besides nuclei, we focused on the evaluation of HNE content in mitochondria with the aim to characterize apoptotis induced by HNE in a cell line p53 and pRB mutated.

The opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs) allow gene expression to be exquisitely regulated through chromatin remodelling. Aberrant transcription due to altered expression or mutation of genes that encode HATs, HDACs or their binding partners, is a key event in the onset and progression of cancer. HDAC inhibitors can reactivate gene expression and inhibit the growth and survival of tumour cells. The remarkable tumour specificity of these compounds, and their potency in vitro and in vivo, underscore the potential of HDAC inhibitors as exciting new agents for the treatment of cancer. The consequence of HDAC inhibition in cultured mammalian cells include reversion of transformed morphology and inhibition of cell proliferation by induction of cell cycle arrest in G1 phase, differentiation and/or apoptosis of tumor cell lines. A number of structurally diverse HDAC inhibitors have been identified, many of which are natural products; among these we have identified 9-hydroxystearic acid (9-HSA), an endogenous long chain lipoperoxidation product (1). We have shown that exposure to 9-HSA results in a significant inhibition of adenocarcinoma colon cancer cells (HT29) proliferation rate, a significant increase of p21 transcription rate, as well as of p21 protein expression level (2). The expression of p21 is induced through a p53-indipendent mechanism, by HDAC1 inhibithion (3). In this work we have isolated histones in order to identify of their acetylation levels, and understand which classes of histones are linked to the 9-HSA induced trancriptional regulation and growth inhibition. Our results have shown that 9-HSA inhibits HDAC activity resulting in the accumulation of acetylated histones. In particular we found that incubation for 6 hours with 9-HSA induced iperacetylation of H4 core histone and H1 linker histone.

HDAC1 (Histone deacetylase) activity is inhibited by 9-hydroxystearic acid (9-HSA) (1,2). HDAC1 inhitors specifically induce differentiation/apoptosis of transformed cells in vitro and suppres tumor growth in vivo. 9-HSA, an endogenous lipid peroxidation product, induces apoptosis characterized by mitochondrial stress, but so far the critical elements of these apoptotic program remain poorly defined. To characterize this apoptotic program in more detail we used a human ostesarcoma cell line p53 w.t. and we studied important elements of the apoptotic response such as p53, Bax and caspase 9. We demonstrate that Bax expression level and caspase 9 activity are critical for apoptosis induced by 9-HSA and that the susceptibility to cell death in response to 9-HSA treatment is regulated by p53 acetylation level.

A combination of spectroscopic techniques, hydrogen/deuterium exchange, and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the topology of the monomeric M*partly folded intermediate of aspartate aminotransferase from Escherichia coli in wild type (WT) as well as in a mutant form in which the highly conserved cis-proline at position 138 was replaced by a trans-alanine (P138A). Fluorescence analysis indicates that, although M* is an off-pathway intermediate in the folding of WT aspartate aminotransferase from E. coli, it seems to coincide with an on-pathway folding intermediate for the P138A mutant. Spectroscopic data, hydrogen/deuterium exchange, and limited proteolysis experiments demonstrated the occurrence of conformational differences between the two M*intermediates, with P138A-M* being conceivably more compact than WT-M*. Limited proteolysis data suggested that these conformational differences might be related to a different relative orientation of the small and large domains of the protein induced by the presence of the cis-proline residue at position 138. These differences between the two M* species indicated that in WT-M* Pro138 is in the cis conformation at this stage of the folding process. Moreover, hydrogen/deuterium exchange results showed the occurrence of few differences in the native N2 forms of WT and P138A, the spectroscopic features and crystallographic structures of which are almost superimposable

Petrosaspongiolide M (PM) is an anti-inflammatory marine metabolite that displays a potent inhibitory activity toward group II and III secretory phospholipase A2 (PLA2) enzymes. The details of the mechanism, which leads to a covalent adduct between PLA2 and γ-hydroxybutenolide-containing molecules such as PM, are still a matter of debate. In this paper the covalent binding of PM to bee venom PLA2 has been investigated by mass spectrometry and molecular modeling. The mass increment observed for the PM-PLA2 adduct is consistent with the formation of a Schiff base by reaction of a PLA2 amino group with the hemiacetal function (masked aldehyde) at the C-25 atom of the PM γ-hydroxybutenolide ring. Proteolysis of the modified PLA2 by the endoprotease LysC followed by HPLC MS analysis allowed us to establish that the PLA2 α-amino terminal group of the lle-1 residue was the only covalent binding site for PM. The stoichiometry of the reaction between PM and PLA2 was also monitored and results showed that even with excess inhibitor, the prevalent product is a 1:1 (inhibitor:enzyme) adduct, although a 2:1 adduct is present as a minor component. The 2:1 adduct was also characterized, which showed that the second site of reaction is located at the ε-amino group of the Lys-85 residue. Similar results in terms of the reaction profile, mass increments, and location of the PLA2 binding site were obtained for manoalide, a paradigm for irreversible PLA2 inhibitors, which suggests that the present results may be considered of more general interest within the field of anti-inflammatory sesterterpenes that contain the γ-hydroxybutenolide pharmacophore. Finally, a 3D model, constrained by the above experimental results, was obtained by docking the inhibitor molecule into the PLA2 binding site through AFFINITY calculations. The model provides an interesting insight into the PM-PLA2 inhibition process and may prove useful in the design of new anti-inflammatory agents that target PLA2 secretory enzymes.

Titin is an exceptionally large protein (M.Wt. approximately 3 MDa) that spans half the sarcomere in muscle, from the Z-disk to the M-line. In the Z-disk, it interacts with alpha-actinin homodimers that are a principal component of the Z-filaments linking actin filaments. The interaction between titin and alpha-actinin involves repeating approximately 45 amino acid sequences (Z-repeats) near the N-terminus of titin and the C-lobe of the C-terminal calmodulin-like domain of alpha-actinin. The conformation of Z-repeat 7 (ZR7) of titin when complexed with the 73-amino acid C-terminal portion of alpha-actinin (EF34) was studied by heteronuclear NMR spectroscopy using (15)N-labeling of ZR7 and found to be helical over a stretch of 18 residues. Complex formation resulted in the protection of one site of preferential cleavage of EF34 at Phe14-Leu17, as determined by limited proteolysis experiments coupled to mass spectrometry measurements. Intermolecular NOEs show Val16 of ZR7 to be positioned close in space to the backbone of EF34 around Phe14. These observations suggest that the mode of binding of ZR7 to EF34 is similar to that of troponin I to troponin C and of peptide C20W to calmodulin. These complexes would appear to represent a general alternative binding mode of calmodulin-like domains to target peptides.