2-DE and MALDI-TOF-MS for a comparative analysis of proteins expressed in different cellular models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disorder characterized by the selective loss of motor neurons from the spinal cord and brain. About 10% of ALS cases are familial (FALS), and in 20% of these cases the disease has been linked to mutations in the Cu,Zn-SOD1 gene. Although the molecular mechanisms causing these forms of ALS are still unclear, evidence has been provided that motor neurons injuries associated with mutant superoxide dismutase (SOD1)-related FALS result from a toxic gain-in-fuction of the mutated enzyme. To understand better the role of these mutations in the pathophysiology of FALS we have compared the pattern of proteins expressed in human neuroblastoma SH-SY5Y cell line with those of cell lines transfected with plasmids expressing the wild-type human SOD1 and the H46R and G93A mutants. 2-DE coupled to MALDI-TOF-MS were the proteomic tools used for identification of differentially expressed proteins. These included cytoskeletal proteins, proteins that regulate energetic metabolism and intracellular redox conditions, and the ubiquitin proteasome system. The proteomic approach allowed to expand the knowledge on the pattern of proteins, with altered expression, which we should focus on, for a better understanding of the possible mechanism involved in mutated-SOD1 toxicity. The cellular models considered in this work have also evidenced biochemical characteristics common to other SOD1-mutated cellular lines connected to the pathogenesis of ALS.     

Specific adsorption of cytochrome C on cardiolipin-glycerophospholipid monolayers and bilayers

In this study, we examined the adsorption of cytochrome c (cyt c) on monolayers and liposomes formed from (i) pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), or cardiolipin (CL) and on (ii) the more thermodynamically stable binary mixtures of POPE/CL (0.8:0.2 mol/mol) and POPC/CL (0.6:0.4 mol/mol). Constant surface pressure experiments showed that the maximum and minimum interactions occurred in the pure CL (anionic phospholipid) and the pure POPE (zwitterion) monolayers, respectively. Observation by atomic force microscopy (AFM) of the images of Langmuir-Blodgett (LB) films extracted at 30 mN m-1 suggests that the different interactions of cyt c with POPE/CL and the POPC/CL monolayers could be due to lateral phase separation occurring in the POPE/CL mixture. The competition between 8-anilino-1-naphthalene sulfonate (ANS) and cyt c for the same binding sites in liposomes that have identical nominal compositions with respect to those of the monolayers was used to obtain binding parameters. In agreement with the monolayer experiments, the most binding was observed in POPE/CL liposomes. All of our observations strongly support the existence of selective adsorption of cyt c on CL, which is modulated differently by different neutral phospholipids (POPE and POPC).     

Mechanism of inhibition of human secretory phospholipase A2 by flavonoids: rationale for lead design

The human secretory phospholipase A2 group IIA (PLA2-IIA) is a lipolytic enzyme. Its inhibition leads to a decrease in eicosanoids levels and, thereby, to reduced inflammation. Therefore, PLA2-IIA is of high pharmacological interest in treatment of chronic diseases such as asthma and rheumatoid arthritis. Quercetin and naringenin, amongst other flavonoids, are known for their anti-inflammatory activity by modulation of enzymes of the arachidonic acid cascade. However, the mechanism by which flavonoids inhibit Phospholipase A2 (PLA2) remained unclear so far. Flavonoids are widely produced in plant tissues and, thereby, suitable targets for pharmaceutical extractions and chemical syntheses. Our work focuses on understanding the binding modes of flavonoids to PLA2, their inhibition mechanism and the rationale to modify them to obtain potent and specific inhibitors. Our computational and experimental studies focused on a set of 24 compounds including natural flavonoids and naringenin-based derivatives. Experimental results on PLA2-inhibition showed good inhibitory activity for quercetin, kaempferol, and galangin, but relatively poor for naringenin. Several naringenin derivatives were synthesized and tested for affinity and inhibitory activity improvement. 6-(1,1-dimethylallyl)naringenin revealed comparable PLA2 inhibition to quercetin-like compounds. We characterized the binding mode of these compounds and the determinants for their affinity, selectivity, and inhibitory potency. Based on our results, we suggest C(6) as the most promising position of the flavonoid scaffold to introduce chemical modifications to improve affinity, selectivity, and inhibition of PLA2-IIA by flavonoids.     

The first open-paddlewheel structures in diruthenium chemistry: examples of intermediate magnetic behaviour between low and high spin in Ru2(5+) species

The reaction of [Ru(2)Cl(O(2)CMe)(DPhF)(3)] (DPhF=N,N'-diphenylformamidinate) with aqueous HCl leads to the substitution of the acetate ligand to give the complex [Ru(2)Cl(2)(DPhF)(3)] (1). Similar reaction of [Ru(2)(O(2)CMe)(DPhF)(3)(H(2)O)]BF(4) with aqueous HBr or HI produces [Ru(2)Br(2)(DPhF)(3)] (2), and [Ru(2)I(2)(DPhF)(3)] (3), respectively. The reaction of 1 with AgBF(4) to form the highly unsaturated unit [Ru(2)(DPhF)(3)](2+), which is isolated as [Ru(2)(BF(4))(DPhF)(3)(H(2)O)]BF(4) (4), and [Ru(2)(MeCN)(2)(DPhF)(3)](BF(4))(2) (5), is also reported. The use of AgNO(3) instead of AgBF(4) leads to [Ru(2)(NO(3))(2)(DPhF)(3)] (6). The magnetic behaviour of complexes 1-4 and 6 is intermediate between high- and low-spin configurations. A relationship between the magnetic behaviour and the visible-near-infrared (Vis-NIR) spectra is apparent. In addition, the crystal structure determinations of 2, 4.THF, and 6, have been carried out. Complexes 1-3, 5 and 6 are the first examples of open-paddlewheel structures in diruthenium chemistry. The BF(4) (-) bridging the metal centres in 4THF is activated and forms very short Ru-F bonds.     

A molecular plug-socket connector

A monocationic plug-socket connector that is composed, at the molecular level, of three components, (1) a secondary dialkylammonium center (CH2NH2+CH2), which can play the role of a plug toward dibenzo[24]crown-8 (DB24C8), (2) a rigid and conducting biphenyl spacer, and (3) 1,4-benzo-1,5-naphtho[36]crown-10 (BN36C10), capable of playing the role of a socket toward a 4,4'-bipyridinium dicationic plug, was synthesized and displays the ability to act as a plug-socket connector. The fluorescent signal changes associated with the 1,5-dioxynaphthalene unit of its BN36C10 portion were monitored to investigate the association of this plug-socket connector with the complementary socket and plug compounds. The results indicate that (1) the CH2NH2+CH2 part of the molecular connector can thread DB24C8 in a trivial manner and (2) the BN36C10 ring of the connector can be threaded by a 1,1'-dioctyl-4,4'-bipyridinium ion only after the CH2NH2+CH2 site is occupied by a DB24C8 ring. The two connections of the three-component assembly are shown to be controlled reversibly by acid/base and red/ox external inputs, respectively. The results obtained represent a key step for the design and construction of a self-assembling supramolecular system in which the molecular electron source can be connected to the molecular electron drain by a molecular elongation cable.     

Diketopiperazines as a tool for the study of transport across the blood-brain barrier (BBB) and their potential use as BBB-shuttles

Here we prepared and evaluated two libraries of mono-N-methylated and di-N-methylated diketopiperazines (DKPs) by parallel artificial membrane permeability assay and immobilized artificial membrane chromatography in order to obtain information on the features that govern the passage of peptidic molecules across the blood-brain barrier (BBB) by passive diffusion. On the basis of the results from these two libraries, we prepared and evaluated several DKP-baicalin and DKP-dopamine constructs. The DKPs or cyclic dipeptide scaffolds can be considered a novel family of brain delivery systems (BBB-shuttles) to transport to the brain drugs and other cargos that cannot cross the BBB unaided.     

13C-detection in RNA bases: revealing structure-chemical shift relationships

The chemical shifts of the unprotonated carbons in the proton-deficient nucleobases of RNA are rarely reported, despite the valuable information that they contain about base-pairing and base-stacking. We have developed 13C-detected 2D-experiments to identify the unprotonated 13C in the RNA bases and have assigned all the base nuclei of uniformly 13C,15N-labeled HIV-2 TAR-RNA. The 13C chemical shift distributions revealed perturbations correlated with the base-pairing and base-stacking properties of all four base-types. From this work, we conclude that the information contained in the chemical shift perturbations within the base rings can provide valuable restraint information for solving RNA structures, especially in conformational averaged regions, where NOE-based information is not available.     

Amido/amine triazacyclononane-based zirconium complexes: syntheses, reactivity, and structures

The reactions of [Zr(NMe2)4]2 with triamido-triazacyclonane ligand precursors, {NH(Ph)SiMe2}3tacn (H3N3[9]N3) and {NH(C6H4F)SiMe2}3tacn (H3N3-F[9]N3), led to the formation of complexes [Zr(NMe2)2{N(Ph)SiMe2}2{NH(Ph) SiMe2}tacn], 1, and [Zr(NMe2)2{N(o-C6H4F)SiMe2}2{NH(o-C6H4F)SiMe2} tacn], 2, where the zirconium is coordinated to two remaining dimethylamido ligands and to a dianionic tacn-based ligand, [{N(Ph')SiMe2}2{NH(Ph')SiMe2}tacn]2-, that formed from deprotonation of two amine pendent arms of the ligands' precursors. The third pendent arm of H3N3[9]N3 and H3N3-F[9]N3 remains neutral and not bonded to the zirconium. Treatment of 1 with NaH led to the synthesis of [Zr(NMe2){N(Ph)SiMe2}2tacn], 3, that results from the cleavage of the N-Si bond of the original neutral pendent arm. Complexes [ZrCl{N(Ph')SiMe2}2tacn] (Ph' = C6H5, 4, and C6H4F, 5) have been obtained by reactions of ZrCl4 with {MN(Ph')SiMe2}3tacn.2THF (M = Li, Na). Reactions of 4 and 5 with LiC triple bond CPh led to the syntheses of [Zr(CCPh){N(Ph')SiMe2}2tacn] (Ph' = C6H5, 6, and C6H4F, 7). The solid-state structure of 3 shows a chiral metal center.