Evidence for the involvement of cytosolic 5'-nucleotidase (cN-II) in the synthesis of guanine nucleotides from xanthosine

In this paper, we show that in vitro xanthosine does not enter any of the pathways known to salvage the other three main natural purine nucleosides: guanosine; inosine; and adenosine. In rat brain extracts and in intact LoVo cells, xanthosine is salvaged to XMP via the phosphotransferase activity of cytosolic 5'-nucleotidase. IMP is the preferred phosphate donor (IMP + xanthosine --> XMP + inosine). XMP is not further phosphorylated. However, in the presence of glutamine, it is readily converted to guanyl compounds. Thus, phosphorylation of xanthosine by cytosolic 5'-nucleotidase circumvents the activity of IMP dehydrogenase, a rate-limiting enzyme, catalyzing the NAD(+)-dependent conversion of IMP to XMP at the branch point of de novo nucleotide synthesis, thus leading to the generation of guanine nucleotides. Mycophenolic acid, an inhibitor of IMP dehydrogenase, inhibits the guanyl compound synthesis via the IMP dehydrogenase pathway but has no effect on the cytosolic 5'-nucleotidase pathway of guanine nucleotides synthesis. We propose that the latter pathway might contribute to the reversal of the in vitro antiproliferative effect exerted by IMP dehydrogenase inhibitors routinely seen with repletion of the guanine nucleotide pools.     

Red wine consumption may affect sperm biology: The effects of different concentrations of the phytoestrogen Myricetin on human male gamete function

Myricetin is a natural flavonoid, particularly enriched in red wines, whose occurrence is widespread among plants. Despite extensive research, the beneficial effects of Myricetin on human health are still controversial. Here, we tested the estrogen‐like effect of the phytoestrogen Myricetin on human ejaculated sperm biology. To this aim, human normozoospermic samples were exposed to increasing concentrations (10 nM, 100 nM, and 1 µM) of Myricetin. Motility, viability, capacitation‐associated biochemical changes (i.e., cholesterol efflux and tyrosine phosphorylation), acrosin activity, as well as glucose utilization and fatty‐acid oxidation (i.e., glucose and lipid metabolism) were all significantly increased by low doses of Myricetin. Importantly, both estrogen receptors α and β (ERs) and phosphatidylinositol‐3‐OH kinase (PI3K)/AKT signaling are activated in the presence of Myricetin since these were both abrogated by specific inhibitors of each pathway. Our results show how Myricetin, through ERs and PI3K/AKT signalings, potentiates sperm function. This effect is dose‐dependent at low concentrations of Myricetin (up to 100 nM), whereas higher amounts do not seem to improve any further sperm motility, viability, or other tested features, and, in some cases, they reduced or even abrogated the efficacy exerted by lower doses. Further studies are needed to elucidate if high levels of Myricetin, which could be attained even with moderate wine consumption, could synergize with endogenous estrogens in the female reproductive tract, interfering with the physiological sperm fertilization process. Mol. Reprod. Dev. © 2012 Wiley Periodicals, Inc.     

Starvation and rapamycin differentially regulate host cell lysosome exocytosis and invasion by Trypanosoma cruzi metacyclic forms

The molecular mechanisms of host cell invasion by T. cruzi metacyclic trypomastigotes (MT), the developmental forms that initiate infection in the mammalian host, are only partially understood. Here we aimed at further identifying the target cell components involved in signalling cascades leading to MT internalization, and demonstrate for the first time the participation of mammalian target of rapamycin (mTOR). Treatment of human epithelial HeLa cells with mTOR inhibitor rapamycin reduced lysosomal exocytosis and MT invasion. Downregulation of phosphatidylinositol 3-kinase and protein kinase C also impaired exocytosis and MT internalization. The recombinant protein based on gp82, the MT surface molecule that mediates cell adhesion/invasion, induced exocytosis in HeLa cells. Such an effect has not previously been attributed to any T. cruzi surface molecule. Rapamycin treatment diminished gp82 binding as well. Cell invasion assays under conditions that promoted lysosome exocytosis, such as 1 h incubation in starvation medium PBS(++) , increased MT invasion, whereas pre-starvation of cells for 1-2 h had an opposite effect. In contrast to MT, invasion of tissue culture trypomastigotes (TCT) increased upon host cell pre-starvation or treatment with rapamycin, a novel finding that discloses quite distinctive features of the two infective forms in a key process for infection.     

Effects of low concentrations of Regorafenib and Sorafenib on human HCC cell AFP,migration, invasion,and growth in vitro

Sorafenib was shown in clinical trial to enhance survival in hepatocellular carcinoma (HCC) patients, but with minimal tumor shrinkage. To correlate several indices of HCC growth at various drug concentrations, HCC cells were grown in various low concentrations of two multikinase inhibitors, Regorafenib (Stivarga) and Sorafenib (Nexavar) and their effects were examined on alpha‐fetoprotein (AFP), cell growth, migration, and invasion. In two AFP positive human HCC cell lines, AFP was inhibited at 0.1–1 µM drug concentrations. Cell migration and invasion were also inhibited at similar low drug concentrations. However, 10‐fold higher drug concentrations were required to inhibit cell growth in both AFP positive and negative cells. To investigate this concentration discrepancy of effects, cells were then grown for prolonged times and sub‐cultured in low drug concentrations and then their growth was re‐tested. The growth in these drug‐exposed cells was found to be slower than cells without prior drug exposure and they were also more sensitive to subsequent drug challenge. Evidence was also found for changes in cell signaling pathways in these slow‐growth cells. Low multikinase inhibitor concentrations thus modulate several aspects of HCC cell biology. J. Cell. Physiol. 228: 1344–1350, 2013. © 2012 Wiley Periodicals, Inc.     

Pyrazolo[1,5-c]quinazoline derivatives and their simplified analogues as adenosine receptor antagonists: Synthesis,structure–affinity relationships and molecular modeling studies

A number of 5-oxo-pyrazolo[1,5-c]quinazolines (series B-1), bearing at position-2 the claimed (hetero)aryl moiety (compounds 1–8) but also a carboxylate group (9–14), were designed as hA₃ AR antagonists. This study produced some interesting compounds endowed with good hA₃ receptor affinity and high selectivity, being totally inactive at all the other AR subtypes. In contrast, the corresponding 5-ammino derivatives (series B-2) do not bind or bind with very low affinity at the hA₃ AR, the only exception being the 5-N-benzoyl compound 19 that shows a hA₃ K_i value in the high μ-molar range. Evaluation of the synthetic intermediates led to the identification of some 5(3)-(2-aminophenyl)-3(5)-(hetero)arylpyrazoles 20–24 with modest affinity but high selectivity toward the hA₃ AR subtype. Molecular docking of the herein reported tricyclic and simplified derivatives was carried out to depict their hypothetical binding mode to our model of hA₃ receptor.     

Taste receptors in the gastrointestinal tract. IV. Functional implications of bitter taste receptors in gastrointestinal chemosensing

Changes in the luminal contents of the gastrointestinal tract modulate gastrointestinal functions, including absorption of nutrients, food intake, and protection against harmful substances. The current notion is that mucosal enteroendocrine cells act as primary chemoreceptors by releasing signaling molecules in response to changes in the luminal environment, which in turn activate nerve terminals. The recent discovery that taste receptors and G protein subunits alpha-gustducin and alpha-transducin, involved in gustatory signal transduction, are expressed in the gastrointestinal mucosa supports the concept of a chemosensory machinery in the gastrointestinal tract. An understanding of luminal sensing processes responsible for the generation of the appropriate functional response to specific nutrients and nonnutrients is of clinical importance since aberrant or unsteady responses to changes in luminal contents might result in disease states ranging from intoxication to feeding disorders and inflammation. The purpose of this theme article is to discuss the functional implications of bitter taste signaling molecules in the gastrointestinal tract deduced by their localization in selected populations of epithelial cells and their relationship with neural pathways responsible for the generation of specific responses to luminal contents.     

FGF2 effects in periosteal fibroblasts bearing the FGFR2 receptor Pro253 Arg mutation

AIM: A growing number of mutations mapped in the receptor gene for fibroblast growth factor have been implicated in several cranial development disorders including the Apert and Crouzon syndromes. The present paper investigated cellular mechanisms underlying Apert phenotype, by analyzing the effects of FGF2 in primary cultures of Apert periosteal fibroblasts carrying the FGFR2 Pro253Arg mutation. RESULTS: FGF2 administration significantly decreased extracellular matrix production in mutant cells by stimulating degradative enzymatic activities. Gene expression analysis revealed that decorin and biglycan, two proteoglycans involved in collagen fibrillogenesis, were more expressed in mutant cells and down-regulated by FGF2. FGF2 receptor binding showed little differences in high affinity receptor counts between mutant and wild-type cells, while we showed for the first time that low affinity receptors are significantly fewer in mutant cells. Differences were found in Crouzon syndrome, where both high and low affinity receptor counts were up-regulated. CONCLUSIONS: The different mutation and low affinity receptor regulation in mutant receptors support the hypothesis that the impact on the activity of the ligand-receptor complex could allow distinct modes of FGF2 activation in Apert and Crouzon syndromes, which interfere with the FGFR2 signalling cascade.