Stabilization of Oxidic Tailings and Contaminated Soils by Calcium Oxyphosphate Addition: The Case of Montevecchio (Sardinia,Italy)

The effectiveness of an in situ heavy metals fixation technique aimed at converting contaminants to low solubility and low bioavail-ability forms, eliminating the risk posed by oxidic tailings and contaminated soils, was investigated. Calcium oxyphosphate salt (Ca(H₂PO₄)₂·H₂O) was used as a stabilizing agent for oxidic tailings and contaminated soils originating from Montevecchio, Sardinia, Italy. Stabilization was effected by mixing the contaminated soil or oxidic tailing sample with calcium oxyphosphate salt at various doses. The effectiveness of stabilization was evaluated by USEPA TCLP standard toxicity testing. Complementary EDTA extraction tests and biological tests using beans Phaseolous vulgaris as plant indicator were carried out. The toxicity of Pb and Cd was reduced below TCLP regulatory limits at calcium oxyphosphate doses higher than 0.7 and 0.2% w/w for soils and tailings, respectively. Lead solubility according to the EDTA test decreased with phosphate dose for both materials tested. Lead uptake by plant leaves and roots from the soil sample decreased with the phosphate addition, while Cd uptake remained almost constant. An adverse effect on plant growth and Zn uptake was observed for calcium oxyphosphate dose up to 1.1% w/w. Based on the results, a remediation scheme for oxidic tailings and contaminated soils is proposed.     

Loss of Tankyrase-mediated destruction of 3BP2 is the underlying pathogenic mechanism of cherubism

Cherubism is an autosomal-dominant syndrome characterized by inflammatory destructive bony lesions resulting in symmetrical deformities of the facial bones. Cherubism is caused by mutations in Sh3bp2, the gene that encodes the adaptor protein 3BP2. Most identified mutations in 3BP2 lie within the peptide sequence RSPPDG. A mouse model of cherubism develops hyperactive bone-remodeling osteoclasts and systemic inflammation characterized by expansion of the myelomonocytic lineage. The mechanism by which cherubism mutations alter 3BP2 function has remained obscure. Here we show that Tankyrase, a member of the poly(ADP-ribose)polymerase (PARP) family, regulates 3BP2 stability through ADP-ribosylation and subsequent ubiquitylation by the E3-ubiquitin ligase RNF146 in osteoclasts. Cherubism mutations uncouple 3BP2 from Tankyrase-mediated protein destruction, which results in its stabilization and subsequent hyperactivation of the SRC, SYK, and VAV signaling pathways.     

Wheat gliadin promotes the interleukin-4-induced IgE production by normal human peripheral mononuclear cells through a redox-dependent mechanism

Increased levels of serum IgE have been described in gliadin-intolerant patients; however, biological mechanisms implicated in this immunoglobulin production remained unknown. In this study, we demonstrated that in vitro crude gliadins and gliadin lysates (Glilys) promoted the IL-4-induced IgE production by human peripheral blood mononuclear cells (PBMC), indicating that the biological process related to gliadin intolerance and/or allergy may lead to IgE production in vivo. It was found that crude gliadin and Glilys potentiated, after 13 days of culture in a dose-dependent manner, IL-4-induced IgE production and, to a lesser extent, the IgG production, while they did not affect IgA or IgM productions. This promoting effect of gliadin and Glilys on the IL-4-induced activation of normal human PBMC was also observed on the early release (2 days) of the soluble fraction of CD23, suggesting its possible involvement in IgE potentiation. The promoting effect of crude gliadin and Glilys appeared to be indirect because they did not modify purified B-lymphocytes IgE production after IL-4 and anti-CD40 monoclonal antibody stimulation.In addition, as revealed by luminol-dependent chemiluminescence, we demonstrated that crude gliadin and Glilys promoted a substantial production of free radicals by normal human PBMC, treated or not with IL-4. This redox imbalance associated with an increased IgE production led us to evaluate the effect of pharmacological antioxidants (N-acetyl-cysteine (NAC) and Cu/Zn-superoxide dismutase (SOD1)) on IgE production by human PBMC. The NAC and the intracellularly delivered SOD1 were found to suppress the IL-4+/-crude gliadin or Glilys-induced IgE production by normal human PBMC. Taken together, these data indicated that gliadin specifically enhanced IL-4-induced IgE production by normal human PBMC, probably by the regulation of redox pathways, and that this 'pro-allergenic' effect could be counteracted by natural antioxidants: thiols and/or vectorized SOD1.     

NMR and molecular dynamics studies of an autoimmune myelin basic protein peptide and its antagonist: structural implications for the MHC II (I-Au)-peptide complex from docking calculations.

Experimental autoimmune encephalomyelitis can be induced in susceptible animals by immunodominant determinants of myelin basic protein (MBP). To characterize the molecular features of antigenic sites important for designing experimental autoimmune encephalomyelitis suppressing molecules, we report structural studies, based on NMR experimental data in conjunction with molecular dynamic simulations, of the potent linear dodecapeptide epitope of guinea pig MBP, Gln74-Lys75-Ser76-Gln77-Arg78-Ser79-Gln80-Asp81-Glu82-Asn83-Pro84-Val85 [MBP(74-85)], and its antagonist analogue Ala81MBP(74-85). The two peptides were studied in both water and Me(2)SO in order to mimic solvent-dependent structural changes in MBP. The agonist MBP(74-85) adopts a compact conformation because of electrostatic interactions of Arg78 with the side chains of Asp81 and Glu82. Arg78 is 'locked' in a well-defined conformation, perpendicular to the peptide backbone which is practically solvent independent. These electrostatic interactions are, however, absent from the antagonist Ala81MBP(74-85), resulting in great flexibility of the side chain of Arg78. Sequence alignment of the two analogues with several species of MBP suggests a critical role for the positively charged residue Arg78, firstly, in the stabilization of the local microdomains (epitopes) of the integral protein, and secondly, in a number of post-translational modifications relevant to multiple sclerosis, such as the conversion of charged arginine residues to uncharged citrullines. Flexible docking calculations on the binding of the MBP(74-85) antigen to the MHC class II receptor site I-A(u) using haddock indicate that Gln74, Ser76 and Ser79 are MHC II anchor residues. Lys75, Arg78 and Asp81 are prominent, solvent-exposed residues and, thus, may be of importance in the formation of the trimolecular T-cell receptor-MBP(74-85)-MHC II complex.     

Drosophila Spag Is the Homolog of RNA Polymerase II-associated Protein 3 (RPAP3) and Recruits the Heat Shock Proteins 70 and 90 (Hsp70 and Hsp90) during the Assembly of Cellular Machineries

The R2TP is a recently identified Hsp90 co-chaperone, composed of four proteins as follows: Pih1D1, RPAP3, and the AAA⁺-ATPases RUVBL1 and RUVBL2. In mammals, the R2TP is involved in the biogenesis of cellular machineries such as RNA polymerases, small nucleolar ribonucleoparticles and phosphatidylinositol 3-kinase-related kinases. Here, we characterize the spaghetti (spag) gene of Drosophila, the homolog of human RPAP3. This gene plays an essential function during Drosophila development. We show that Spag protein binds Drosophila orthologs of R2TP components and Hsp90, like its yeast counterpart. Unexpectedly, Spag also interacts and stimulates the chaperone activity of Hsp70. Using null mutants and flies with inducible RNAi, we show that spaghetti is necessary for the stabilization of snoRNP core proteins and target of rapamycin activity and likely the assembly of RNA polymerase II. This work highlights the strong conservation of both the HSP90/R2TP system and its clients and further shows that Spag, unlike Saccharomyces cerevisiae Tah1, performs essential functions in metazoans. Interaction of Spag with both Hsp70 and Hsp90 suggests a model whereby R2TP would accompany clients from Hsp70 to Hsp90 to facilitate their assembly into macromolecular complexes.     

AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis

Oxidative stress is a principal cause of DNA damage, and mechanisms to repair this damage are among the most highly conserved of biological processes. Oxidative stress is also used by phagocytes to attack bacterial pathogens in defence of the host. We have identified and characterised two apurinic/apyrimidinic (AP) endonuclease paralogues in the human pathogen Neisseria meningitidis. The presence of multiple versions of DNA repair enzymes in a single organism is usually thought to reflect redundancy in activities that are essential for cellular viability. We demonstrate here that these two AP endonuclease paralogues have distinct activities in DNA repair: one is a typical Neisserial AP endonuclease (NApe), whereas the other is a specialised 3'-phosphodiesterase Neisserial exonuclease (NExo). The lack of AP endonuclease activity of NExo is shown to be attributable to the presence of a histidine side chain, blocking the abasic ribose-binding site. Both enzymes are necessary for survival of N. meningitidis under oxidative stress and during bloodstream infection. The novel functional pairing of NExo and NApe is widespread among bacteria and appears to have evolved independently on several occasions.     

Cellular oligomerization of alpha-synuclein is determined by the interaction of oxidized catechols with a C-terminal sequence

The mechanisms that govern the formation of alpha-synuclein (alpha-syn) aggregates are not well understood but are considered a central event in the pathogenesis of Parkinson's disease (PD). A critically important modulator of alpha-syn aggregation in vitro is dopamine and other catechols, which can prevent the formation of alpha-syn aggregates in cell-free and cellular model systems. Despite the profound importance of this interaction for the pathogenesis of PD, the processes by which catechols alter alpha-syn aggregation are unclear. Molecular and biochemical approaches were employed to evaluate the mechanism of catechol-alpha-syn interactions and the effect on inclusion formation. The data show that the intracellular inhibition of alpha-syn aggregation requires the oxidation of catechols and the specific noncovalent interaction of the oxidized catechols with residues (125)YEMPS(129) in the C-terminal region of the protein. Cell-free studies using novel near infrared fluorescence methodology for the detection of covalent protein-ortho-quinone adducts showed that although covalent modification of alpha-syn occurs, this does not affect alpha-syn fibril formation. In addition, oxidized catechols are unable to prevent both thermal and acid-induced protein aggregation as well as fibrils formed from a protein that lacks a YEMPS amino acid sequence, suggesting a specific effect for alpha-syn. These results suggest that inappropriate C-terminal cleavage of alpha-syn, which is known to occur in vivo in PD brain or a decline of intracellular catechol levels might affect disease progression, resulting in accelerated alpha-syn inclusion formation and dopaminergic neurodegeneration.     

Classification and gradient analysis of the beech forest vegetation of the southern Rodopi (northeast Greece)

Pure and mixed beech forest vegetation of the southern Rodopi range (northeast Greece) was studied using 614 relevés and multivariate analyses (TWINSPAN and DCA). Classification of the relevés resulted in 12 vegetation units, 8 of which were ranked as associations or communities and the rest as subcommunities and variants. DCA diagrams of relevés and taxa indicated that floristic differentiation was attributed mainly to factors such as altitude (affecting temperature and humidity), soil nutrient content and substrate type (affecting physical and chemical soil properties). Differential taxa of vegetation units were chosen based on their phi coefficient values, which were calculated from three different percentage synoptic tables that corresponded to three ranks (ecological groups, associations and communities, and subcommunities and variants) of floristic differentiation. The calculation of phi coefficient on the basis of relative constancy of taxa helps to overcome the problem of the dependence of fidelity values on the number of relevés per vegetation unit and to facilitate the better investigation of the floristic differentiation even of rare vegetation units represented by a small number of relevés. Furthermore, the calculation of fidelity values for different hierarchical levels enables a more detailed and thorough investigation of the floristic differentiation of the vegetation units.