Novel potent and highly selective human A(3) adenosine receptor antagonists belonging to the 4-amido-2-arylpyrazolo[3,4-c]quinoline series: molecular docking analysis and pharmacological studies

The study of novel 2-arylpyrazolo[3,4-c]quinolin-4-(hetero)arylamides, designed as human (h) A(3) adenosine receptor antagonists, is reported. The new derivatives are endowed with nanomolar hA(3) receptor affinity and high selectivity versus hA(1), hA(2A) and hA(2B) receptors. Among the (hetero)aroyl residues introduced on the 4-amino group, the 2-furyl and 4-pyridyl rings turned out to be the most beneficial for hA(3) affinity (K(i)=3.4 and 5.0nM, respectively). An intensive molecular docking study to a rhodopsin-based homology model of the hA(3) receptor was carried out to obtain a 'structure-based pharmacophore model' that proved to be helpful for the interpretation of the observed affinities of the new hA(3) pyrazoloquinoline antagonists.     

BDNF prevents NMDA‐induced toxicity in models of Huntington's disease: the effects are genotype specific and adenosine A2A receptor is involved

NMDA receptor‐mediated excitotoxicity is thought to play a pivotal role in the pathogenesis of Huntington's disease (HD). The neurotrophin brain‐derived neurotrophic factor (BDNF), which is also highly involved in HD and whose effects are modulated by adenosine A_2ARs, influences the activity and expression of striatal NMDA receptors. In electrophysiology experiments, we investigated the role of BDNF toward NMDA‐induced effects in HD models, and the possible involvement of A_2ARs. In corticostriatal slices from wild‐type mice and age‐matched symptomatic R6/2 mice (a model of HD), NMDA application (75 μM) induced a transient or a permanent (i.e., toxic) reduction of field potential amplitude, respectively. BDNF (10 ng/mL) potentiated NMDA effects in wild‐type, while it protected from NMDA toxicity in R6/2 mice. Both effects of BDNF were prevented by A_2AR blockade. The protective effect of BDNF against NMDA‐induced toxicity was reproduced in a cellular model of HD. These findings may have very important implications for the neuroprotective potential of BDNF and A_2AR ligands in HD.     

Health status of the lizard Podarcis siculus (Rafinesque-Schmaltz, 1810) subject to different anthropogenic pressures

The terrestrial lizard Podarcis siculus is the most abundant reptile in Italy, where is considered a ubiquitous species. This lizard is widely distributed from the islands to the Apennines, from cultivated fields to anthropized areas such as gardens and city parks. For this reason, these animals are exposed to extensive physical and chemical stresses, as well as to the possibility of coming into contact with industrial pollutants and substances used in agricultural practices. Here, we review the health status of lizard specimens inhabiting natural non-anthropized areas and fields devoted to organic farming, considering the condition of (1) liver, representing the main detoxifying organ, directly influenced by feeding, and (2) gonads, essential for reproduction and, therefore, for the survival of the species. The morphological and biomolecular condition of these organs was then compared with those obtained from lizard specimens experimentally treated with nonylphenol, a co-formulant of many insecticides and plant protection products widely used in conventional farming, known to have harmful estrogenic effects. Taken together, data demonstrate that lizards inhabiting manured soil are in good health status and show a regular morphology of liver, testis, and ovary. Animals are found to be less exposed to the toxic heavy metals cadmium and lead if compared with specimens collected in areas not devoted to agriculture, but probably more exposed to vehicular traffic. However, manure, as well as nonylphenol, exerts a xeno-estrogenic effect, particularly evident in male specimens, more sensitive to estrogenic contamination.     

Structure and nucleotide sequence of Euphorbia characias copper/TPQ-containing amine oxidase gene

A cDNA encoding for a copper containing amine oxidase has been isolated and sequenced from young leaves of Euphorbia characias, a perennial mediterranean shrub. A single long open reading frame of 2068 pb encodes a protein composed of 653 amino acids with a molecular mass of about 74 kDa. A putative 24-aminoacid signal peptide precedes the sequence of the mature protein, with characteristics of a secretion signal peptide. Alignments of Euphorbia amine oxidase cDNA nucleotide sequence with that of amine oxidase from the seedlings of the pulses lentil, pea, and chickpea reveal several conserved regions, especially in the C-terminus, with a homology 90%–97%. The near 5 region shows several insertions, deletions, and different nucleotide sequence with ca. 60% homology. The enzyme contains 1%–2% carbohydrate deduced by deglycosylation experiments. Five cysteine residues are present in the deduced aminoacid sequence with a single disulfide bridge as judged by titration with cysteine reagents.     

Mechanism-based inactivators of plant copper/quinone containing amine oxidases

Copper/quinone amine oxidases contain Cu(II) and the quinone of 2,4,5-trihydroxyphenylalanine (topaquinone; TPQ) as cofactors. TPQ is derived by post-translational modification of a conserved tyrosine residue in the protein chain. Major advances have been made during the last decade toward understanding the structure/function relationships of the active site in Cu/TPQ amine oxidases using specific inhibitors. Mechanism-based inactivators are substrate analogues that bind to the active site of an enzyme being accepted and processed by the normal catalytic mechanism of the enzyme. During the reaction a covalent modification of the enzyme occurs leading to irreversible inactivation. In this review mechanism-based inactivators of plant Cu/TPQ amine oxidases from the pulses lentil (Lens esculenta), pea (Pisum sativum), grass pea (Lathyrus sativus) and sainfoin (Onobrychis viciifolia,) are described. Substrates forming, in aerobiotic and in anaerobiotic conditions, killer products that covalently bound to the quinone cofactor or to a specific amino acid residue of the target enzyme are all reviewed.     

Platelet‐Rich Plasma Increases Growth and Motility of Adipose Tissue‐Derived Mesenchymal Stem Cells and Controls Adipocyte Secretory Function

Adipose tissue‐derived mesenchymal stem cells (Ad‐MSC) and platelet derivatives have been used alone or in combination to achieve regeneration of injured tissues. We have tested the effect of platelet‐rich plasma (PRP) on Ad‐MSC and adipocyte function. PRP increased Ad‐MSC viability, proliferation rate and G1‐S cell cycle progression, by at least 7‐, 2‐, and 2.2‐fold, respectively, and reduced caspase 3 cleavage. Higher PRP concentrations or PRPs derived from individuals with higher platelet counts were more effective in increasing Ad‐MSC growth. PRP also accelerated cell migration by at least 1.5‐fold. However, PRP did not significantly affect mature adipocyte viability, differentiation and expression levels of PPAR‐γ and AP‐2 mRNAs, while it increased leptin production by 3.5‐fold. Interestingly, PRP treatment of mature adipocytes also enhanced the release of Interleukin (IL)‐6, IL‐8, IL‐10, Interferon‐γ, and Vascular Endothelial Growth Factor. Thus, data are consistent with a stimulatory effect of platelet derivatives on Ad‐MSC growth and motility. Moreover, PRP did not reduce mature adipocyte survival and increased the release of pro‐angiogenic factors, which may facilitate tissue regeneration processes. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc. J. Cell. Biochem. 116: 2408–2418, 2015. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

Cannabidiol reduces Aβ-induced neuroinflammation and promotes hippocampal neurogenesis through PPARγ involvement

Peroxisome proliferator-activated receptor-γ (PPARγ) has been reported to be involved in the etiology of pathological features of Alzheimer's disease (AD). Cannabidiol (CBD), a Cannabis derivative devoid of psychomimetic effects, has attracted much attention because of its promising neuroprotective properties in rat AD models, even though the mechanism responsible for such actions remains unknown. This study was aimed at exploring whether CBD effects could be subordinate to its activity at PPARγ, which has been recently indicated as its putative binding site. CBD actions on β-amyloid-induced neurotoxicity in rat AD models, either in presence or absence of PPAR antagonists were investigated. Results showed that the blockade of PPARγ was able to significantly blunt CBD effects on reactive gliosis and subsequently on neuronal damage. Moreover, due to its interaction at PPARγ, CBD was observed to stimulate hippocampal neurogenesis. All these findings report the inescapable role of this receptor in mediating CBD actions, here reported.     

Characterization of Large Unilamellar Vesicles as Models for Studies of Lipid Peroxidation Initiated by Azocompounds

The aim of this work was to characterize large unilamellar vesicles (LUVETs) prepared by a hand-driven extrusion device in order to use them for studies of lipid peroxidation and antioxidant activity. Vesicle structure and size were examined by electron microscopy. Lipid and antioxidant content was determined before and after the extrusion procedure. Then LUVETs were subjected to autoxidation initiated by both the lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile) and the water-soluble 2,2'-azobis(2-amidinopropane hydrochloride) azocompounds. The results demonstrated that: i) LUVETs prepared with lipid concentrations ranging between 25 and 150 mM were essentially unilamellar and reasonably homogeneous, with an average diameter of 90 nm; ii) the phospholipid, cholesterol and antioxidant amounts retained by filters were about 10-15%; iii) LUVETs were suitable for autoxidation studies initiated by the water-soluble azocompound both in the absence and presence of antioxidants. The lipid-soluble azocompound could be used only at low concentrations and its vesicle content had to be determined since part of the initiator was not incorporated into the lipid bilayer. These data suggest that LUVETs seem to be recommended for studies of lipid peroxidation and antioxidant activity.     

Association of Genetic Variation in Adaptor Protein APPL1/APPL2 Loci with Non-Alcoholic Fatty Liver Disease

The importance of genetics and epigenetic changes in the pathogenesis of non alcoholic fatty liver disease (NAFLD) has been increasingly recognized. Adiponectin has a central role in regulating glucose and lipid metabolism and controlling inflammation in insulin-sensitive tissues and low adiponectin levels have been linked to NAFLD. APPL1 and APPL2 are adaptor proteins that interact with the intracellular region of adiponectin receptors and mediate adiponectin signaling and its effects on metabolism. The aim of our study was the evaluation of a potential association between variants at APPL1 and APPL2 loci and NAFLD occurrence. The impact on liver damage and hepatic steatosis severity has been also evaluated. To this aim allele frequency and genotype distribution of APPL1- rs3806622 and -rs4640525 and APPL2-rs 11112412 variants were evaluated in 223 subjects with clinical diagnosis of NAFLD and compared with 231 healthy subjects. The impact of APPL1 and APPL2 SNPs on liver damage and hepatic steatosis severity has been also evaluated. The minor-allele combination APPL1-C/APPL2-A was associated with an increased risk of NAFLD (OR = 2.50 95% CI 1.45–4.32; p<0.001) even after adjustment for age, sex, body mass index, insulin resistance (HOMA-IR), triglycerides and adiponectin levels. This allele combination carrier had higher plasma alanine aminotransferase levels (Diff = 15.08 [7.60–22.57] p = 0.001) and an increased frequency of severe steatosis compared to the reference allele combination (OR = 3.88; 95% CI 1.582–9.531; p<0.001). In conclusion, C-APPL1/A-APPL2 allele combination is associated with NAFLD occurrence, with a more severe hepatic steatosis grade and with a reduced adiponectin cytoprotective effect on liver.