Settlement of a stable wolf pack in a highly anthropic area of Pisan hills: Relationship with animal husbandry and hunting in a human-wolf coexistence perspective.

Anthropic areas play a pivot role for main wolf conservation challenges. Wolf presence in the higher Pisan hills has been well documented while wolf settlement in the lower Pisan hills is still uncertain. In this study, long-term information on wolf presence in a highly anthropic area of the lower Pisan hills was collected by using non-invasive monitoring techniques. Furthermore, both the relationship of this predator with human activity and the impact of hunting on wolf presence have been investigated. The results obtained indicate the presence of a stable and reproductive wolf pack composed by both Italian wolf and hybrids individuals in the municipalities of Crespina Lorenzana and Casciana Terme Lari. A high impact of wolf on livestock was recorded in this area since no prevention systems were adopted by farmers. Wolf appears not to have a negative impact on wild boar population. Similarly, wild boar drive hunting does not appear to affect the wolf pack presence in the area. Thereby wolf may play a key role as controller of wild population. Prevention strategies improvement becomes instrumental to promote wolf-human coexistence. Further investigation to monitor pack hybridization level and turnover and to assess the impact of packs on wild population and livestock in anthropic areas is desirable.

Searching for an absolute kinetic scale of antioxidant activity against lipid peroxidation.

The inhibition properties of a number of antioxidants against peroxidation, started by a 2,2'-azobis[2-(2-imidazolin-2-yl)propane] radical initiator, of linoleic acid in sodium dodecyl sulfate micelles, have been determined in terms of oxygen consumption by a Clark electrode in an oxygen-tight cell. For the 31 antioxidants investigated at variable concentrations, the experimental results well fit the kinetic equation for competitive reactions. The ratio between the initial rates, monitored in the absence and in the presence of antioxidants, depends linearly on their concentration. From the slopes of these straight lines, an absolute scale of inhibition properties of the lipid peroxidation can be devised. Furthermore, the little difference of the time of complete oxygen consumption on concentration of different antioxidants has been found, indicating a restricted difference towards chemical structure and stoichiometric ratio. Some considerations regarding the mechanisms of inhibition of the lipid peroxidation in micelles, in view of bibliographic data, have been made.

Genetic predisposition to breath-hold diving-induced hemoptysis: Preliminary study.

INTRODUCTION: Breath-hold diving-induced hemoptysis (BH-DIH) has been reported in about 25% breath-hold divers (BHD) and is characterized by dyspnea, coughing, hemoptysis and chest pain. We investigated whether eNOS G894T, eNOS T786C and ACE insertion/deletion I/D genetic variants, are possible BH-DIH risk factors. METHODS: 108 experienced healthy instructor BHDs with the same minimum requirements (102 male, six female; mean age 43.90 ± 7.49) were studied. We looked for different eNOS G894T, eNOS T786C and ACE insertion/ deletion genetic variants between BH-DIH-positive and BH-DIH-negative subjects to identify the variants most frequently associated with BH-DIH. RESULTS: At least one BH-DIH episode was reported by 22.2% of subjects, while 77.7% never reported BH-DIH. The majority of BH-DIH-positive subjects showed eNOS G894T (p = 0.001) and eNOS-T786C (p = 0.001) genotype "TT" (high-risk profile). Prevalence of BH-DIH was higher in subjects with eNOS G894T TT genotype (50%) than in subjects with GT (9.5%, p < 0.001) and GG (24%, (p = 0.0002) genotype (low-risk profile). Similar results were observed for eNOS T786C: BH-DIH prevalence was higher in the TT genotype (41.2%) group than in the CT (15.4%, p < 0.001) and CC genotype (9.1%, p < 0.001) groups. BH-DIH prevalence was significantly higher in subjects showing ACE ID genotype (34.5%) than II (0%, p < 0.001) and DD (10.5%, p = 0.0002). Of the ACE "II" genotype group, 100% never developed BH-DIH. DISCUSSION: eNOS-G894T, eNOS-T786C and ACE influence NO availability and regulation of peripheral vascular tone and blood flow. Different genetic variants of eNOS-G894T, eNOS-T786C and ACE appear significantly related to the probability to develop BH-DIH (p < 0.001).

Cooperative nucleophilic-electrophilic organocatalysis by ionic liquids.

The anionic and the cationic partners of ionic liquids may act cooperatively and independently as nucleophilic and electrophilic catalysts. This ambiphilic propensity was demonstrated by kinetically discriminating the contributions of the anion (nucleophilic catalyst) and of the cation (electrophilic catalyst) to the solvent-free Baylis-Hillman dimerization of cyclohexenone catalysed by ionic liquids.

Metal-promoted synthesis of amidines containing the model nucleobases 1-methylcytosine and 9-methyladenine.

The amidine complexes cis-[L(2)PtNH==C(R){1-MeCy(-2H)}]NO(3) (R = Me, 1a; Ph, 1b, Me(3)C, 1c; Ph(2)(H)C, 1d) and cis-[L(2)PtNH==C(R){9-MeAd(-2H)}]NO(3) (R = Me, 2a; Ph, 2b; Me(3)C, 2c; Ph(2)(H)C, 2d), are formed when cis-[L(2)Pt(µ-OH)](2)(NO(3))(2) (L = PPh(3)) reacts with 1-methylcytosine (1-MeCy) and 9-methyladenine (9-MeAd) in solution of MeCN, PhCN, Me(3)CCN and Ph(2)(H)CCN. Reaction of 1a,b and 2a,b with HCl affords the protonated amidines [NH(2)==C(R){1-MeCy(-H)}]NO(3) (R = Me, 3a; Ph, 3b) and [NH(2)==C(R){9-MeAd(-H)}]NO(3) (R = Me, 4a; Ph, 4b) and cis-(PPh(3))(2)PtCl(2) in quantitative yield. Treatment of 3b and 4b with NaOH allows the isolation of the neutral benzimidamides NH(2)-C(Ph){1-MeCy(-2H)} (5b) and NH(2)-C(Ph){9-MeAd(-2H)} (6b). In the solid state 3b shows a planar structure with the hydrogen atom on N(4) cytosine position involved in a strong H-bond with the NO(3)(-) ion. Intermolecular H-bonds between the oxygen of the cytosine ring and one of the H atoms of the amidine-NH(2) group allow the dimerization of the molecule. A detailed analysis of the spectra of 3b in DMF-d(7) at -55 °C indicates the presence of an equilibrium between the species [NH(2)==C(R){1-MeCy(-H)}]NO(3) and [NH(2)==C(R){1-MeCy(-H)}](2)(NO(3))(2), exchanging with trace amounts of water at 25 °C. [(15)N,(1)H]-HMBC experiments for 5b and 6b indicate that the amino tautomer H(2)N-C(Ph){nucleobase(-2H)}, is the only detectable in solution and such structure has been confirmed in the solid state. The reaction of 5b and 6b with cis-L(2)Pt(ONO(2))(2) (L = PPh(3)), in chlorinated solvents, determines the immediate appearance of a pale yellow colour due to the coordination of the neutral amidine, likely in its imino form HN==C(Ph){nucleobase(-H)}, to give the adducts cis-[L(2)PtNH==C(Ph){nucleobase(-H)}](2+). In fact, addition of "proton sponge" leads to the immediate deprotonation of the amidine ligand with formation of the starting complexes 1b and 2b.

The reaction of primary aromatic amines with alkylene carbonates for the selective synthesis of bis-N-(2-hydroxy)alkylanilines: the catalytic effect of phosphonium-based ionic liquids.

At T≥ 140 °C, different primary aromatic amines (pX-C(6)H(4)NH(2); X = H, OCH(3), CH(3), Cl) react with both ethylene- and propylene-carbonates to yield a chemoselective N-alkylation process: bis-N-(2-hydroxyalkyl)anilines [pX-C(6)H(4)N(CH(2)CH(R)OH)(2); R = H, CH(3)] are the major products and the competitive formation of carbamates is substantially ruled out. At 140 °C, under solventless conditions, the model reaction of aniline with ethylene carbonate goes to completion by simply mixing stoichiometric amounts of the reagents. However, a class of phosphonium ionic liquids (PILs) such as tetraalkylphosphonium halides and tosylates turn out to be active organocatalysts for both aniline and other primary aromatic amines. A kinetic analysis monitored by (13)C NMR spectroscopy, shows that bromide exchanged PILs are the most efficient systems, able to impart a more than 8-fold acceleration to the reaction. The reactions of propylene carbonate take place at a higher temperature than those of ethylene carbonate, and only in the presence of PIL catalysts. A mechanism based on the Lewis acidity of tetraalkylphosphonium cations and the nucleophilicity of halide anions has been proposed to account for both the reaction chemoselectivity and the function of the catalysts.

Synthesis, structure-activity relationship and crystallographic studies of 3-substituted indolin-2-one RET inhibitors.

The synthesis, structure-activity relationships (SAR) and structural data of a series of indolin-2-one inhibitors of RET tyrosine kinase are described. These compounds were designed to explore the available space around the indolinone scaffold within RET active site. Several substitutions at different positions were tested and biochemical data were used to draw a molecular model of steric and electrostatic interactions, which can be applied to design more potent and selective RET inhibitors. The crystal structures of RET kinase domain in complex with three inhibitors were solved. All three compounds bound in the ATP pocket and formed two hydrogen bonds with the kinase hinge region. Crystallographic analysis confirmed predictions from molecular modelling and helped refine SAR results. These data provide important information for the development of indolinone inhibitors for the treatment of RET-driven cancers.

Ionic liquids made with dimethyl carbonate: solvents as well as boosted basic catalysts for the michael reaction.

This article describes 1) a methodology for the green synthesis of a class of methylammonium and methylphosphonium ionic liquids (ILs), 2) how to tune their acid-base properties by anion exchange, 3) complete neat-phase NMR spectroscopic characterisation of these materials and 4) their application as active organocatalysts for base-promoted carbon-carbon bond-forming reactions. Methylation of tertiary amines or phosphines with dimethyl carbonate leads to the formation of the halogen-free methyl-onium methyl carbonate salts, and these can be easily anion-exchanged to yield a range of derivatives with different melting points, solubility, acid-base properties, stability and viscosity. Treatment with water, in particular, yields bicarbonate-exchanged liquid onium salts. These proved strongly basic, enough to efficiently catalyse the Michael reaction; experiments suggest that in these systems the bicarbonate basicity is boosted by two orders of magnitude with respect to inorganic bicarbonate salts. These basic ionic liquids used in catalytic amounts are better even than traditional strong organic bases. The present work also introduces neat NMR spectroscopy of the ionic liquids as a probe for solute-solvent interactions as well as a tool for characterisation. Our studies show that high catalytic efficacy of functional ionic liquids can be achieved by integrating their green synthesis, along with a fine-tuning of their structure. Demonstrating that ionic liquid solvents can be made by a truly green procedure, and that their properties and reactivity can be tailored to the point of bridging the gap between their use as solvents and as catalysts.

Characterization of compound 584, an Abl kinase inhibitor with lasting effects.

BACKGROUND: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosome-positive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584). DESIGN AND METHODS: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotrans-planted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor. RESULTS: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC(50): 8 nM) and in cell-based assays (IC(50): 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC(50): 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib. CONCLUSIONS: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former.

Chiral polycyclic ketones via desymmetrization of dihaloolefins.

3-Chloronorbornenone (R)-1a (98% ee) was obtained from trichloronorbornene 5 in two steps by the in situ generation of dichloronorbornadiene 2a with t-BuOK and desymmetrization with (-)-ephedrine, followed by hydrolysis with PPTS. The generality of this desymmetrization with (-)-ephedrine was tested with dibromonorbornadiene 2c and other substituted dichloronorbornadienes.

From the Apennines to the Alps: colonization genetics of the naturally expanding Italian wolf (Canis lupus) population.

Wolves in Italy strongly declined in the past and were confined south of the Alps since the turn of the last century, reduced in the 1970s to approximately 100 individuals surviving in two fragmented subpopulations in the central-southern Apennines. The Italian wolves are presently expanding in the Apennines, and started to recolonize the western Alps in Italy, France and Switzerland about 16 years ago. In this study, we used a population genetic approach to elucidate some aspects of the wolf recolonization process. DNA extracted from 3068 tissue and scat samples collected in the Apennines (the source populations) and in the Alps (the colony), were genotyped at 12 microsatellite loci aiming to assess (i) the strength of the bottleneck and founder effects during the onset of colonization; (ii) the rates of gene flow between source and colony; and (iii) the minimum number of colonizers that are needed to explain the genetic variability observed in the colony. We identified a total of 435 distinct wolf genotypes, which showed that wolves in the Alps: (i) have significantly lower genetic diversity (heterozygosity, allelic richness, number of private alleles) than wolves in the Apennines; (ii) are genetically distinct using pairwise F(ST) values, population assignment test and Bayesian clustering; (iii) are not in genetic equilibrium (significant bottleneck test). Spatial autocorrelations are significant among samples separated up to c. 230 km, roughly correspondent to the apparent gap in permanent wolf presence between the Alps and north Apennines. The estimated number of first-generation migrants indicates that migration has been unidirectional and male-biased, from the Apennines to the Alps, and that wolves in southern Italy did not contribute to the Alpine population. These results suggest that: (i) the Alps were colonized by a few long-range migrating wolves originating in the north Apennine subpopulation; (ii) during the colonization process there has been a moderate bottleneck; and (iii) gene flow between sources and colonies was moderate (corresponding to 1.25-2.50 wolves per generation), despite high potential for dispersal. Bottleneck simulations showed that a total of c. 8-16 effective founders are needed to explain the genetic diversity observed in the Alps. Levels of genetic diversity in the expanding Alpine wolf population, and the permanence of genetic structuring, will depend on the future rates of gene flow among distinct wolf subpopulation fragments.

Development and exploitation of CK2 inhibitors.

A number of quite specific and fairly potent inhibitors of protein kinase CK2, belonging to the classes of condensed polyphenolic compounds, tetrabromobenzimidazole/triazole derivatives and indoloquinazolines are available to date. The structural basis for their selectivity is provided by a hydrophobic pocket adjacent to the ATP/GTP binding site, which in CK2 is smaller than in the majority of other protein kinases due to the presence of a number of residues whose bulky side chains are generally replaced by smaller ones. Consequently a doubly substituted CK2 mutant V66A,I174A is much less sensitive than CK2 wild type to these classes of inhibitors. The most efficient inhibitors both in terms of potency and selectivity are 4,5,6,7-tetrabromo-1H-benzotriazole, TBB (Ki = 0.4 microM), the TBB derivative 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, DMAT (Ki = 0.040 microM), the emodin related coumarinic compound 8-hydroxy-4-methyl-9-nitrobenzo[g]chromen-2-one, NBC (Ki = 0.22 microM) and the indoloquinazoline derivative ([5-oxo-5,6-dihydroindolo-(1,2a)quinazolin-7-yl]acetic acid), IQA (Ki = 0.17 microM). These inhibitors are cell permeable as judged from ability to block CK2 in living cells and they have been successfully employed, either alone or in combination with CK2 mutants refractory to inhibition, to dissect signaling pathways affected by CK2 and to identify the endogenous substrates of this pleitropic kinase. By blocking CK2 these inhibitors display a remarkable pro-apoptotic efficacy on a number of tumor derived cell lines, a property which can be exploited in perspective to develop antineoplastic drugs.

Analysis of pellets found in the north area of the Asiago plateau (Vicenza, Italy), a locality of the 1914-1918 World War.

Pellets of unknown material contained in an aluminium cylinder were found in the north area of the Asiago plateau (Vicenza), a locality of the First World War (1914-1918). Elemental analysis, infrared, chromatography and NMR experiments indicate that the main product is pentaerythritol tetranitrate (PETN). This substance was probably an igniting primer used by Austro-Hungarian (A.U.) military engineering. Hypothesis of medical use of the pellets can be disregarded while it seems improbable the use of this explosive by German Army during the Second War World.

Internal motions in a fulleropyrrolidine tertiary amide with axial chirality.

The kinetic parameters for topomerization around the N-CO bond and enantiomerization around the C-CO bond in N-1-naphthoyl fulleropyrrolidine 1 and N-1-naphthoyl pyrrolidine 2 have been determined by dynamic NMR (line shape simulation and selective inversion transfer). The DeltaS(not =) values are negligible. The DeltaH# value for topomerization of 1 is smaller with respect to that of 2 by 4.3 kcal mol(-1) (explained by the electron-withdrawing effect of fullerene) and the value for enantiomerization is greater by 1.4 kcal mol(-1) (explained by the greater rigidity of the fulleropyrrolidine ring, as confirmed by ab initio analyses).

AFLP: a useful tool for biodiversity conservation and management.

The access to new molecular markers is not always possible for all researchers, due to limited laboratory and economical resources. A new technique, called AFLP, seems to be a simple and robust method to obtain a large number of variable molecular markers. In this article I show the results of AFLP analysis in three different conservation projects requiring different level of genetic variability. I also compare the multi-locus AFLP results with single-locus markers (microsatellites) and haploid organellar marker (mtDNA) sequences. The results suggest that the AFLP technique could be very useful in a wide range of conservation studies.

Efficient cyclotrimerization of bicyclic vic-bromostannylalkenes promoted by copper(I) thiophen-2-carboxylate.

Copper(I) thiophen-2-carboxylate was successfully employed in the trimerization of [2.2.1] bicyclic vic-bromotrimethyltin olefins (in their racemic composition), bearing different functionalities, to invariably obtain almost quantitative yields of the syn and anti tris-annelated benzenes. The two isomers come in different ratios, smaller than or equal to the statistical 1:3 ratio, depending on the steric hindrance opposed by the functionalities. In the case of enantiopure (3-bromo-4,7,7-trimethylbicyclo[2.2.1]hept-2-en-2-yl)trimethylstannane, the 1:9 ratio found with Cu(NO(3))(2).3H(2)O increases to 1:6.

Aerobic biodegradation of aliphatic polyethoxylates: an 1H-nuclear magnetic resonance spectroscopy investigation.

Aerobic biodegradation tests of three blends representative of the most commonly marketed aliphatic alcohol polyethoxylates (AE) and a commercial polyethylene glycols (PEG) mixture were run under standard test conditions (Organization for Economic Cooperation and Development, Paris, France [OECD] 301 E protocol). The test liquors were investigated using a 400-MHz proton-nuclear magnetic resonance (1H-NMR) spectrometer in order to elucidate the biodegradation mechanism. Diagnostic signals for both linear and branched AE homologs were identified by bidimensional homocorrelated spectroscopy (COSY) and by double quantum filter correlated spectroscopy (DQFCOSY). The 1H-NMR allowed individually monitoring the fate of the alkyl and polyethoxyl fragments of the parent compounds and distinguishing between oxidative and nonoxidative polyethoxyl depolymerization, which could not be performed by other reported techniques such as high-peformance liquid chromatography (HPLC) or mass spectrometry (MS) or FAB spectroscopy. The AE biodegradation time profiles showed that under the test conditions, both alkyl and polyethoxyl chains of the linear and oxo-AE were biodegraded quite readily. The removal of the multibranched AE was slower when compared to that of linear and oxo-AE, while PEG exhibited a time profile characterized by a biodegradation rate significantly slower than that of PEG released by the microbial attack of linear and oxo-AE. In the case of linear AE, the alkyl chain was biodegraded much faster than the polyethoxyl chain, while in the case of oxo- and multibranched AE, both the alkyl and the polyethoxyl chains were biodegraded at similar rates.

Oxyfunctionalization of Non-Natural Targets by Dioxiranes. 3.(1) Efficient Oxidation of Buckminsterfullerene C(60) with Methyl(trifluoromethyl)dioxirane.

By employing methyl(trifluoromethyl)dioxirane (1b), the stepwise oxyfunctionalization of C(60) can be carried out with high conversions (>90%) under mild conditions (0 degrees C); the products have been compared with those produced by the oxidation of C(60) with m-chloroperoxybenzoic acid. Along with the previously characterized oxide C(60)O, a wider set of higher oxidation products is obtained by using 1b; among these, regioisomeric dioxides C(60)O(2) are isolated in good overall yield (40%). One of the dioxides is predominant (yield 23%), corresponding to a C(s)()-symmetry dioxide previously well characterized and presenting the epoxide functionalities in close proximity over the 6:6 ring junctions. The oxidation with dioxirane 1b also produces sufficient quantities of trioxides, so that mixtures of C(60)O(3) regioisomers can be isolated. The main trioxide fraction was found amenable to spectroscopic characterization; the (13)C NMR spectra indicates that the sample consists of two possible regioisomers, one having C(s)(), and the other C(2) symmetry. In both, the three epoxide rings are assembled over 6:6 ring junctions and in close proximity to each other; this shows that, in the ensuing sequential O-transfers from the dioxirane to the fullerene framework, the 6:6 carbon-carbon double bonds adjacent to an existing epoxide functionality are more easily oxidized. The whole of the spectroscopic data indicate that the fullerene core remains intact and no rupture of the cage occurs following oxidation at the trioxide level.

Central to Axial Transfer of Chirality in Menthone or Camphor-Derived 2,2'-Biphenols.

A study aimed at defining a molecular arrangement where a chiral fragment derived from menthone or camphor transfers its central chirality to a 2,2'-biphenol residue, inducing an axial chirality, is reported. The menthol or isoborneol groups are attached at the two benzylic positions at 3,3' in order to maximize efficiency in practical applications. A reliable and high-yielding procedure for the synthesis of such C(2)-symmetric molecules substituted at the 3,3'-positions has been developed. The procedure entails Mannich condensation with paraformaldehyde and morpholine, protection of the hydroxylic functions, chlorination, metalation, and addition to (-)-menthone and (+)-camphor. The use of samarium diiodide is essential in the latter step for optimum selectivity and efficiency. The tetrols exhibit intramolecular hydrogen bonding between phenolic and alcoholic hydroxy functions within each monomeric unity, so that they retain their rotational freedom. NOEDS and COSY experiments show that the tetrols are present in more than one rotamer. The tetrols react with tetrachlorosilane to afford siloxanes as pure diastereoisomers, showing that the metal is able to induce preferential helicity at the biphenyl residue; i.e., the central chirality of menthol or isoborneol auxiliary is totally transfered to the axial chirality of the biphenyl. The configurations could be determined by NOEDS and heterocorrelated HMQC experiments. Remarkably, while the menthol derivative induces total M helicity, the camphor induces complementary P helicity. These results suggest that these tetrols may be useful as ligands in catalysts for asymmetric synthesis.