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The soft acid CdII is an accumulative toxic metal ion which is able to substitute for the essential borderline ZnII ion in many zinc enzymes. Nuclear magnetic resonance (NMR) spectroscopy was used to assess the coordination ability of two ligands, an amine-bearing bis-kojic acid: 6'-(2-(diethylamino)ethylazanediyl)bis(methylene)bis(5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) [1,2] and a hydroxypiridinone derivative: 5-hydroxy-2-(hydroxymethyl)pyridin-4(1H)-one [3], for their use as potential chelating molecules towards CdII ions in detoxification treatments. A combination of 1D, 2D total correlation spectroscopy (TOCSY), heteronuclear single quantum coherence spectroscopy (HSQC) and rotating-frame Overhauser effect spectroscopy (ROESY) experiments was used to assign the signals of both free and metal-bound ligands, as previously reported for similar systems [4-6]. The metal-ligand system was studied at physiological pH and different temperature values. Competition experiments with essential ZnII ions were also performed.

Several metals have toxic actions on nerve cells and neurobehavioral functioning playing a role in the genesis and development of many neurodegenerative diseases. In particular manganese(II) is a well known neurotoxic metal as it accumulates in basal ganglia inducing Manganism, a pathology with common manifestations with Parkinson's disease. YPK9 gene (Yeast PARK9; also known as YOR291W) encoded protein can protect cells from manganese poisoning. In fact YPK9 is a non-essential yeast gene predicted by sequence to encode a transmembrane P-type transport ATPase involved in metal coordination and transportation. Mutations in the human homolog of YPK9, ATP13A2/PARK9, have been linked to genetic forms of early onset Parkinsonism. We tested the binding ability of Mn(II) and other divalent metal ions (Cu(II), Zn(II), Ni(II)) with several peptide sequences from YPK9 with particular focus on highly conserved sequences from yeast to human. The work was carried out at different pH values and ligand/metal molar ratios by means of potentiometric and spectroscopic techniques (multidimensional and heteronuclear NMR and UV-visible), in order to evaluate and compare the coordination propensity of such fragments with Mn(II) and the other metal probes selected, with the purpose of sheding a light on the protective properties of YPK9 in Manganese-induced Parkinsonism.

Chelation therapy is a consolidated medical procedure used primarily to hinder the effects of toxic metal ions on human tissues. Its application spans a broad spectrum of disorders, ranging from acute metal intoxication to genetic metal-overload. The use of chelating agents is compromised by a number of serious side effects, mainly attributable to perturbed equilibrium of essential metal ion homeostasis and dislocation of complexed metal ions to dangerous body sites. For this reason, chelation therapy has been limited to specific critical and otherwise untreatable conditions and needs to be monitored within an appropriate clinical context. An alarming issue today is that fraudsters use the term “chelation therapy” to take advantage of and make profit from people with tragic health problems. We believe that scientists working in this field have the corollary obligation to deter these frauds and to inform the scientific community of the possible side effects and complications of chelation therapy. This duty is all the more important if we consider the detrimental and even life threatening consequences that can occur in subjects with no clear clinical and laboratory evidence of metal intoxication. The aim of this communication is to present how this “false chelation therapy” developed and in which diseases it is currently applied.

Chelation therapy is used for the treatment of metal intoxication in humans. Selectivity towards the target metal ion is one important characteristic of the chelating agent. In the frame of our research of chelating agents for iron and aluminium, we synthesized five new ligands (Figure 1), and studied their behavior toward the trivalent metal ions. L4, L5, L6 and L8 were found to be excellent ligands for the coordination of Fe3+ and Al3+. We are presenting here a study on the same ligands with the two essential bivalent metal ions, Zn2+ and Cu2+. The results of spectrophotometric, potentiometric, and NMR measurements performed to determine the equilibrium formation constants will be presented. The speciation of the complexes with the trivalent metal ions in presence of endogenous zinc and copper will be discussed.

In the attempt to set up a useful methodology for the investigation of burned human remains in archaeological, anthropological and forensic fields, we decided to compare the most common protocols for the study of bone bioapatites (Fourier Transform Infrared spectroscopy, FT-IR, and X-ray Diffraction, XRD) to those deriving from the application of X-ray scattering techniques using synchrotron light. In this way, we expect to take advantage of the wider and more dynamic qualities of such a valuable tool in order to examine a higher number of samples in a very short time compared to the “traditional” techniques, meanwhile assessing its applicability in the archaeological field.

A variety of metals are essential trace elements but can reach localized toxic concentrations through various disease processes or environmental exposures and have been implicated as having a role in neurodegeneration. In particular, chronic inorganic manganese exposure causes selective toxicity to the nigrostriatal dopaminergic system, resulting in a Parkinsonian-like neurological condition known as Manganism. YPK9 gene (Yeast PARK9; also known as YOR291W) encodes a transmembrane P-type transport ATPase presumably involved in metal coordination and transportation, though its substrate specificity still remains unknown. Mutations in the human homolog of YPK9, PARK9 (ATP13A2), have been linked to genetic forms of early onset parkinsonism. Recently a strong genetic interaction between YPK9 and another Parkinson's disease protein, α-synuclein, has been evidenced in multiple model systems, indicating a crucial role for YPK9 in manganese detoxification in yeast and a specific protecting effect against manganese poisoning [1,3]. With the purpose to shed light on the protective property of YPK9 in Manganese-induced Parkinsonism, we tested the binding ability of Mn(II) and other divalent cations (Cu(II), Zn(II)) towards several peptide sequences from YPK9, with a particular focus on highly conserved sequences from yeast to human. The work was carried out at different pH values and ligand/metal molar ratios by means of potentiometric and spectroscopic techniques (multidimensional and heteronuclear NMR and UV-visible), in order to evaluate and compare the coordination propensity of such fragments with Mn(II) and the other metal probes selected [4,5].

Nickel allergy is the most frequent cause of contact hypersensitivity (burning, redness, itching, swelling and even blisters) in industrialized countries, with 30% of population being affected. Contact allergy is commonly induced by nickel ions present in nickel-containing jewelry such as rings and earrings, as well as in nickel-containing cellular telephones. Ni(II) seems to trigger an inflammatory response by activating human Toll-like-Receptor 4 (hTLR4) [1-4]. Species-specific activation, as in this case, requires distinct sequence motifs that are present in humans but not in mouse, a species not sensitive to nickel-induced allergies. A sequence containing three histidine residues, H431, and the non-conserved H456 and H458, localized in the C-terminus, could be identified as the specific region of human TLR4 responsible for nickel responses. It has been proposed that the imidazole side chain of the histidine residues H456 and H458 may provide a potential binding site for this metal because they are located at an optimal distance to interact with Ni(II) ions, whereas H431 is located further apart. The aim of our research was to verify the possibility of metal binding to the sequence containing the three histidines supposedly involved in nickel response. The chosen segment was the 32aa peptide FQH431SNLKQMSEFSVFLSLRNLIYLDISH456TH458TR, which was studied in order to understand both its binding properties and the thermodynamic stability of its metal complexes. Formation equilibria of Ni(II) complexes have been investigated in aqueous solution and in a wide pH range. Protonation and complex-formation constants have been potentiometrically determined; complex-formation models and species stoichiometry have been checked by means of UV-Vis absorption and CD spectroscopy and investigation through multidimensional and eteronuclear NMR spectroscopy. The predominant species for a 1:1 peptide/Ni(II) molar ratio was obtained at physiological pH and showed an effective binding of the metal to the target sequence.

Contact allergy, commonly induced by nickel, is the most frequent cause of contact hypersensitivity in industrialized countries, with 30% of population being affected. Ni(II) seems to trigger an inflammatory response by activating human Tool-like-Receptor 4 (hTLR4). Species-specific activation, as in this case, required distinct sequence motifs that are present in human but not in mouse, a species not sensitive to nickel-induced allergies. The specific region of human TLR4 responsible for nickel responses could be a sequence containing three histidine residues, H431, and the non- conserved H456 and H458, localized in the C-terminus. It has been proposed that the imidazole side chains of the histidine residues H456 and H458 provide a potential binding site for nickel because they were located at an optimal distance to interact with Ni(II) ions, whereas H431 was further apart. We decided to verify the possibility of metal binding to FQH431SNLKQMSEFSVFLSLRNLIYLDISH456TH458TR sequence, containing the three histidines supposedly involved in nickel response, in order to study the binding properties of the peptide fragment and on the thermodynamic stability of its metal complexes. Formation equilibria of Ni(II) complexes have been investigated in aqueous solution and in a wide pH range. Protonation and complex-formation constants have been potentiometrically determined; complex-formation models and species stoichiometry have been checked by means of UV-Vis absorption and CD spectroscopy and investigation through NMR is currently being carried out. The predominant species for a 1:1 peptide/Ni(II) molar ratio was obtained at physiological pH and showed an effective binding of the metal to the target sequence.

Follicular Fluid (FF) provides a special environment to the oocyte during its maturation in vivo. The FF is derived from the sanguineous plasma and secretions, synthesised in the follicle wall that contain a large variety of metabolites (1). These metabolites are probably involved in the physiology of the oocytes (1). The chemical composition of follicular fluids is important because it is an indicator of the secretory activities and metabolism of follicular cells and thus could be related to the follicular quality. It could also provide a useful indication of the oocyte growth and maturation (2). High Resolution Nuclear Magnetic Resonance (NMR) spectroscopy provides a unique tool for studying metabolites. Initially, NMR spectroscopy was used mainly in biomedicine but it is found now in many physiological applications (3). As the NMR spectroscopy provides opportunities for obtaining qualitative and quantitative data from body fluids, it was hypothesized that this technique could provide information on mammals’ follicular fluid and on intrafollicular changes that occur during follicular growth and ovulation. As some of these changes are probably of crucial importance for oocyte developmental competence, a better knowledge of the mammals’ follicular fluid composition by 1H NMR analysis should help to resolve some of the problems encountered during in vitro procedures in the mammals. The characterization of the chemical composition of mammals follicular fluids, namely sheep, cattle, mare and pork, and the study of the changes observed during follicular growth and maturation using NMR spectroscopy will be presented. FF samples were collected from antral follicle of different dimensions. One-dimensional (1D) 1H experiments (CPMG, DOSY) were obtained for all the FF samples. In addition, several two dimensional (2D) (homo and heteronuclear) experiments (DQF-COSY, TOCSY, JRES, 1H-13C HSQC) were performed to aid in the assignment of the signals and in the identification of the metabolites in FF. A direct evaluation of the lipids, carbohydrates and metabolites were obtained from the combination of the 1D and 2D NMR experiments.

In the frame of our research interest on kojic acid derivatives as powerful chelators for the trivalent iron and aluminium cations [1-4], we have designed, synthesized, and characterized the new ligand 6,6'-(((2-(diethylamino)ethyl)azanediyl)bis(methylene))bis(5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one), L9. In this communication will be present the study on protonation constants and on the complex formation equilibria with iron and aluminium and with the bivalent essential metal ions, zinc and copper. X-ray structures of the ligand and of some of its metal complexes will be also presented.

La Malattia di Parkinson (MP) è una patologia neurodegenerativa le cui cause non sono state ancora comprese appieno. Per questo motivo è detta anche sindrome idiopatica, cioè senza cause note o identificabili, sebbene alcuni tipi di MP possano avere una causa genetica o post-traumatica, e diversi fattori di rischio come l’esposizione ad alcuni pesticidi. Recentemente è emerso che anche l’esposizione al manganese (come capita ai minatori o ai saldatori) può causare una sindrome simile alla MP (Parkinsonismo), ed è stata scoperta una correlazione tra cause genetiche e ambientali attraverso lo studio della mutazione di una proteina chiamata Park9.1 Successivamente, una ricerca sul gene di un lievito, YPK9, che è molto simile al PARK9 umano, ha rivelato che la cancellazione di questo gene conferisce al lievito una maggiore sensibilità alla crescita in presenza diversi cationi divalenti, suggerendo che la proteina Ypk9 possa giocare un ruolo nel sequestro di metalli pesanti nelle loro forme divalenti.2 Allo stesso modo, una mutazione sul PARK9 potrebbe esporre l’uomo all’azione di queste specie cationiche. In quest’ottica, abbiamo scelto dei piccoli fragmenti della proteina Ypk9 che includono sequenze interessanti per un potenziale legame con i metalli, altamente conservate in un grosso numero di organismi e collocate in porzioni della proteina facilmente accessibili ai metalli. Abbiamo inizialmente studiato il loro comportamento nei confronti di cationi divalenti come il manganese e lo zinco, usando tecniche NMR mono- e bidimensionali, insieme alla spettroscopia EPR.3 Durante lo svolgimento di questo studio abbiamo iniziato inoltre quello relativo alla coordinazione del Cu(II), e da una serie di misure potenziometriche e spettroscopiche è emerso che anche questo metallo è in grado di legarsi efficacemente alla proteina.

Parkinson Disease (PD) is a neurodegenerative pathology whose causes have not yet been fully clarified. In this perspective, we have chosen short fragments of Ypk9 protein that include interesting sequences for metal binding and are highly conserved in a number of different organisms, located in domains of the protein readily accessible to the metals. We studied their behaviour towards divalent cations such as manganese and zinc, using NMR mono- and bidimensional techniques and EPR spectroscopy. As this study was going on, we also started the investigation on Cu(II) coordination, showing by a series of potentiometric and spectroscopic measurements that this metal too is able to effectively bind the chosen sequence. Here we would like to report our latest findings.

It has been found that anionic species of metal salts can lead to different geometries and stoichiometries in the synthesis of coordination compounds. Therefore, using the same cation but different polyatomic anion groups such as oxalate, sulfate, acetate, oxamate etc. [1], it is possible to obtain metal complexes with various structural and stoichiometric features upon coordination. In fact, these anionic groups can behave themselves, generally, as mono-, bidentate and bridgedbidentate ligands towards metal ions. In the last years, with the aim to design a multi-dentate ligand capable of supporting simultaneous coordination to three copper atoms and to mimic the multicopper active sites of blue copper oxidases (e.g., laccase, ascorbate oxidase and ceruloplasmin), we focused our studies on the synthesis and structural characterization of copper(II) complexes of compartmental acyclic bis(salicylhydrazone) ligands derived from iminoand methyl-iminodiacetic acid, finding a different behaviour using copper(II) sulfate, perchlorate and acetate salts. Thus, we have obtained, with a 1:3 (ligand-to-metal) molar ratio, a trinuclear coordination polymer from Cu(II) perchlorate [2], a sulfato-bridged hexanuclear dimer [3] and a mononuclear complex using Cu(II) acetate [4]. Microbiological investigations showed a good activity of the sulfato and perchlorato complexes. Studies concerning DNA binding of these compounds are now in progress.

It is known that rich repeat domains in peptides can be of interest as the models for the study of molecular phenomena related to metal ion binding in proteins involved in neurodegenerative disorders. Imbalances in transition metal ions are assumed to contribute to the conversion of the multi-histidinic amyloid β-peptide (Aβ) from its soluble form to an amyloidogenic form, and to Aβ deposition. Of these ions, it has been reported that manganese binding to PrP is detrimental and causes a conformational change in the protein, suggesting that manganese binding could potentially play a role in prion disease progression in vivo. It appears that PrP is less stable on binding manganese and quickly converts to a misfolded form. The binding of manganese to PrP potentially results in the conversion of the protein to an abnormal isoform with properties reminiscent of PrPsc. In particular, although PrP can bind the same number of manganese atoms as of copper atoms, the resulting protein becomes proteinase resistant, forms fibrils and loses function.[1,2] Regarding cobalt, a novel low-affinity binding site for Co(II) was discovered between PrP residues 104 and 114, with residue His111 being the key amino acid for coordinating Co(II).[3] Thus, despite the interest in manganese and cobalt binding to PrP, a thorough analysis of the interaction of both metals with proteins related to brain pathies has not yet been reported. The (T1R2S3R4S5H6T7S8E9G10)3 fragment from Cap43 protein, which is induced by metal ions, is characterized by a decarepeat domain comprising three decapeptide units with one histidine and one glutamate residue in each repeat. Therefore the study of the interaction of the 30-aminoacid peptide from Cap43 protein with metal ions can contribute to the understanding of the crucial role of multi-imidazol and glutamate sites in the protein coordination processes and the possible role of divalent metal ions in the pathogenesis of prion disease and other related protein pathies.[4-8] Here we present our recent results on the Cobalt(II) and Manganese(II) complexes of terminally protected mono- and multi-histidine-glutamate peptides studied by combination of potentiometric measurements and spectroscopic techniques (NMR, UV-Vis and EPR). Metal complexation induces important structural changes with the C-terminal portion of the ligand, constraining it to leave its disordered conformation and promoting side chain orientation. Our results give rise to a molecular model of the induced structure for the peptides complexed with cobalt and manganese.

Nickel compounds are well known as human carcinogens, though the molecular events that are responsible for this are not well understood. It has been proposed that a crucial element in the mechanism of carcinogenesis is the binding of Ni(II) ions within the cell nucleus. It is known that DNA polymer binds Ni(II) only weakly, leaving the proteins of the cell nucleus as the likely Ni(II) targets. Being histone proteins the most abundant among them, they can be considered the primary sites for nickel binding. Here we describe the interactions of nickel with histone H4, core tetramer (H3-H4)2 and several peptide fragments which have been selected as the candidates for specific binding sites in the histone octamer. The results allowed us to propose several mechanisms of nickel induced damage resulting from metal coordination, including structural changes of histone proteins, as well as nucleobase oxidation and sequence-specific histone hydrolysis. The aim of the present work is to provide a comprehensive overview of literature dealing with nickel coordination to histone proteins and its link with nickel involvement in toxicity and carcinogenicity.

Parkinson Disease (PD) is a neurodegenerative pathology whose causes have not been yet fully clarified. For this reason it is also called an idiopathic (with no known or identifiable causes) syndrome, although some PD types can have a genetic or a post-traumatic origin, and different risk factors like exposure to some pesticides. Recently it emerged that also exposure to manganese (i.e. in welders or miners) can cause a PD-like syndrome (Parkinsonism), and a connection between genetic and environmental causes of Parkinson's disease has been discovered: a genetic interaction between two Parkinson's disease genes (alpha-synuclein and PARK9, alias ATP13A2) was found, and it was determined that the PARK9 protein can protect cells from manganese poisoning. Shortly after, a study on a yeast gene, YPK9, which is 58% similar and 38% identical in its amino acid sequence to human PARK9, revealed that deletion of this gene confers sensitivity for growth for cadmium, manganese, nickel and selenium, suggesting that the YPK9 protein may play a role in the sequestration of divalent heavy metal ions. In the same way, a mutation on PARK9 may expose humans to these cations, especially to manganese. In this perspective, we have chosen short fragments of YPK9 protein that included interesting sequences for metal binding and studied their behaviour towards divalent cations such as manganese and calcium, using NMR mono- and bidimensional techniques and EPR spectroscopy. If metal binding were clearly assessed in the yeast analogue, we could get a hint of what may happen in humans. Here we would like to present our latest findings.

E' noto come l'accumulo dei metalli nel cervello possa portare, o quantomeno contribuire, allo sviluppo di malattie neurodegenerative, attraverso meccanismi che solo ora cominciano a essere lentamente chiariti. La malattia di Parkinson, cosiddetta “idiopatica” in quanto non presenta alcuna causa apparente, è stata messa di recente in correlazione con l'esposizione o l'avvelenamento da manganese. Studi epidemiologici condotti da diverse università degli Stati Uniti hanno evidenziato come gli abitanti in zone urbane con alte concentrazioni di questo metallo avessero una probabilità di sviluppare il Parkinson quasi due volte più alta rispetto agli abitanti in zone meno inquinate, o inquinate da altri metalli (quali ad esempio il rame). Altri studi apparsi di recente in letteratura correlano l'esposizione al manganese con alcune modificazioni di un gene legato alla sinucleina, proteina presente con diverse funzioni in tutte le malattie neurodegenerative. Lo studio è stato effettuato su una proteina di un lievito la YPK9, al 58% simile e al 38% uguale all'analoga umana PARK9, la cui mutazione causa appunto lo sviluppo di una forma ereditaria di Parkinson. Silenziando il gene YPK9 nei lieviti si è notato che in assenza della relativa proteina questi mostravano disturbi nella crescita se sottoposti all'azione di diversi metalli, mentre in presenza del manganese la crescita era particolarmente ridotta. Veniva quindi dimostrata l'azione protettiva della YPK9 nei confronti dei cationi bivalenti, specialmente del manganese. Pare dunque possibile che una modifica sull'analogo umano, il PARK9, sia in grado di inficiare i normali meccanismi con cui il nostro organismo si protegge da ioni metallici dannosi, quali il manganese, e dando il via a una serie di processi che portano allo sviluppo della malattia neurodegenerativa. Abbiamo pertanto voluto verificare l'effettiva propensione di tale proteina a interagire con ioni Mn(II), selezionando sulla sequenza della YPK9 dei frammenti promettenti per il legame con il metallo e investigando la possibilità di una interazione efficace di questi frammenti con diversi cationi bivalenti, tra cui appunto il manganese, ma anche il calcio e lo zinco. I risultati preliminari, ottenuti attraverso alcune tecniche spettroscopiche quali l'NMR mono- e bidimensionale e l'EPR, verranno esposti in questa comunicazione.