Potentiation of temozolomide antitumor effect by purine receptor ligands able to restrain the in vitro growth of human glioblastoma stem cells

Glioblastoma multiforme (GBM), the most common and aggressive brain tumor in humans, comprises a population of stem-like cells (GSCs) that are currently investigated as potential target for GBM therapy. Here, we used GSCs isolated from three different GBM surgical specimens to examine the antitumor activity of purines. Cultured GSCs expressed either metabotropic adenosine P1 and ATP P2Y receptors or ionotropic P2X₇ receptors. GSC exposure for 48 h to 10–150 μM ATP, P2R ligand, or to ADPβS or MRS2365, P2Y₁R agonists, enhanced cell expansion. This effect was counteracted by the PY₁R antagonist MRS2500. In contrast, 48-h treatment with higher doses of ATP or UTP, which binds to P2Y_2/4R, or 2′(3′)-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), P2X₇R agonist, decreased GSC proliferation. Such a reduction was due to apoptotic or necrotic cell death but mostly to growth arrest. Accordingly, cell regrowth and secondary neurosphere formation were observed 2 weeks after the end of treatment. Suramin, nonselective P2R antagonist, MRS1220 or AZ11645373, selective A₃R or P2X₇R antagonists, respectively, counteracted ATP antiproliferative effects. AZ11645373 also abolished the inhibitory effect of Bz-ATP low doses on GSC growth. These findings provide important clues on the anticancer potential of ligands for A₃R, P2Y₁R, and P2X₇R, which are involved in the GSC growth control. Interestingly, ATP and BzATP potentiated the cytotoxicity of temozolomide (TMZ), currently used for GBM therapy, enabling it to cause a greater and long-lasting inhibitory effect on GSC duplication when readded to cells previously treated with purine nucleotides plus TMZ. These are the first findings identifying purine nucleotides as able to enhance TMZ antitumor efficacy and might have an immediate translational impact.     

The effect of chitosan and other polycations on tight junction permeability in the human intestinal Caco-2 cell line(1)

Chitosan is a polycationic compound widely employed as dietary supplement and also present in pharmaceutical preparations. Although it has been approved for human consumption, its possible side effects have not been widely investigated and the available data in the literature are still controversial. Several polycationic substances have been shown to affect tight junction permeability in epithelial cell models in vitro. In this study we have compared the effects of chitosan and other polycations (polyethylenimine, poly-L-lysines of different molecular weights) on the integrity of tight junctions and of the actin cytoskeleton in the human intestinal Caco-2 cell line. We have measured trans-epithelial electrical resistance and paracellular passage of the extracellular marker inulin, and we have localized F-actin and tight junctional proteins (ZO1 and occludin) in cell monolayers treated with various concentrations of each polycation. Fluorescent poly-L-lysines were also employed to determine their association with the cell monolayer. Our results indicate that all polycations investigated are able to induce a reversible increase in tight junction permeability. This effect is concentration and energy dependent, affected by the extracellular concentration of divalent cations (calcium, magnesium and manganese) and it is associated with morphological changes in the F-actin cytoskeleton, as well as in the localization of tight junctional proteins. Chitosan, in particular, was the only cationic polymer that displayed an irreversible effect on tight junctions at the highest concentration tested (0.01%). These results indicate that oral ingestion of chitosan may have more widespread health effects by altering intestinal barrier function, thus allowing the entrance into the circulation of potentially toxic and/or allergenic substances.     

Leaf mineral concentrations of Erica arborea, Juniperus communis and Myrtus communis growing in the proximity of a natural CO2 spring

Leaf mineral concentrations of co‐occurring Erica arborea, Juniperus communis and Myrtus communis were measured at bimonthly intervals throughout a year in a natural CO₂ spring and in a nearby control site with similar soil chemistry in a Mediterranean environment. There were different responses to the elevated [CO₂] (c. 700 μL L^?1) of the spring site plants depending on the element and the species. In the CO₂ spring site K, Ca, Mg, Mn, Al, Fe, and Ti leaf concentrations and the ratio C/N showed significant greater values in at least one or two of the three species. Leaf S concentration were greater in all three species. Leaf concentrations of N, Sr, Co, and B were lower in at least one or two species, and those of C and Ba were lower in all the three studied species near the CO₂ spring. P, Na, Zn, Si, Cu, Ni, Cr, Pb, Mo, V and Cd leaf concentrations and the specific leaf area (SLA, measured in Myrtus communis) did not show any consistent or significant pattern in response to the elevated [CO₂] of the spring site. There was a slight trend towards maximum concentrations of most of these elements during autumn–winter and minimum values during the spring season, especially in Myrtus communis. Multivariate principal component analyses based on the leaf elemental concentrations clearly differentiated the two sites and the three species. Lower concentrations at the spring site were not the result of a dilution effect by increased structural or nonstructural carbon. In contrast to most experimental studies of CO₂ enrichment, mainly conducted for short periods, several of these elements had greater concentrations in the CO₂ spring site. Nutrient acclimation and possible causes including decreased nutrient export, increased nutrient uptake capacity, photosynthetic down‐regulation, Mediterranean water stress, and higher H₂S concentration in the spring site are discussed.     

Characterization of β-glucosidase activity in yeasts of oenological origin

I. ROSI, M. VINELLA AND P. DOMIZIO. 1994. Three hundred and seventeen strains representing 20 species of yeasts were screened for the presence of β-glucosidase activity. All of the strains of the species Debaryomyces castellii, Deb. hansenii, Deb. polymorphus, Kloeckera apiculata and Hansenula anomala showed β-glucosidase activity, but only one of 153 strains of Saccharomyces cerevisiae. The other species behaved differently, depending upon the strain. The strains that hydrolysed arbutin were checked to localize the β-glucosidase activity. A strain of Deb. hansenii exhibited the highest exocellular activity and some wall-bound and intracellular activity. The β-glucosidase synthesis from this yeast was enhanced by aerobic conditions of growth, was repressed by high glucose concentration (9%) and occurred during exponential growth. The optimum conditions for enzymatic preparations of Deb. hansenii were between pH 4.0 and 5.0 and 40C. A high concentration of ethanol and glucose did not reduce the ezymatic activity. The enzymatic preparations of Deb. hansenii released monoterpenols and other alcohols from a grape glycoside extract.     

Split of HLA-DRw2 into subtypic specificities closely correlated to two HLA-D products

Two B-lymphocyte-specific human alloantisera were studied, PA59 and 51.23. They identify two new HLA-DR alleles, which are both subtypic to HLA-DRw2. Moreover, they are closely correlated with two HLA-D products, Dw2 and tb24 (tb24 is a new specificity described by our group). Thus, DRw2 can be split into two subtypic specificities that have been named TO60 and TO61, which appear more strongly correlated with HLA-D antigens. Absorption studies demonstrated cytotoxicity-negative, absorption-positive (CYNAP) reactions, and cross-reactive groups of antibodies.     

A new susceptibility locus for migraine with aura in the 15q11-q13 genomic region containing three GABA-A receptor genes

Migraine is the most common type of chronic episodic headache. Several population-based family studies have suggested a strong genetic predisposition to migraine, especially migraine with aura (MA). Although several susceptibility loci have been identified, none of the numerous studies performed to date have led to the identification of a gene responsible for the more common forms of migraine. GABA-A receptors and their modulator sites seem to be involved in the pathophysiological events that underlie migraine. We report on clinical and molecular data from a total of 10 families with MA, in which MA segregates as an autosomal dominant trait and presents with homogeneous clinical features. After excluding linkage with the known candidate loci, we used a functional candidate approach and genotyped these families with markers from the 15q11-q13 genomic region, which contains the genes encoding GABA-A receptor subunits. Evidence of linkage was obtained with a parametric two-point linkage analysis (maximum LOD score of 5.56 at a recombination fraction of 0.001 for marker GABRB3) and was supported by multipoint analysis (maximum LOD score of 6.54 between markers D15S113 and D15S1019). The critical region spanned 3.6 Mb. These results provide the basis for further investigation of the hypothesized relationship between a GABA-A receptor dysfunction and migraine.     

Caterpillars of Euphydryas aurinia (Lepidoptera: Nymphalidae) feeding on Succisa pratensis leaves induce large foliar emissions of methanol

A major new discovery made in the last decade is that plants commonly emit large amounts and varieties of volatiles after damage inflicted by herbivores, and not merely from the site of injury. However, analytical methods for measuring herbivore-induced volatiles do not usually monitor the whole range of these compounds and are complicated by the transient nature of their formation and by their chemical instability. Here we present the results of using a fast and highly sensitive proton transfer reaction-mass spectrometry (PTR-MS) technique that allows simultaneous on-line monitoring of leaf volatiles in the pptv (pmol mol(-1)) range. The resulting on-line mass scans revealed that Euphydryas aurinia caterpillars feeding on Succisa pratensis leaves induced emissions of huge amounts of methanol--a biogeochemically active compound and a significant component of the volatile organic carbon found in the atmosphere--and other immediate, late and systemic volatile blends (including monoterpenes, sesquiterpenes and lipoxygenase-derived volatile compounds). In addition to influencing neighboring plants, as well as herbivores and their predators and parasitoids, these large emissions might affect atmospheric chemistry and physics if they are found to be generalized in other plant species.     

A flavanone from Baccharis retusa (Asteraceae) prevents elastase-induced emphysema in mice by regulating NF-κB,oxidative stress and metalloproteinases

BackgroundPulmonary emphysema is characterized by irreversible airflow obstruction, inflammation, oxidative stress imbalance and lung remodeling, resulting in reduced lung function and a lower quality of life. Flavonoids are plant compounds with potential anti-inflammatory and antioxidant effects that have been used in folk medicine. Our aim was to determine whether treatment with sakuranetin, a flavonoid extracted from the aerial parts of Baccharis retusa, interferes with the development of lung emphysema.MethodsIntranasal saline or elastase was administered to mice; the animals were then treated with sakuranetin or vehicle 2 h later and again on days 7, 14 and 28. We evaluated lung function and the inflammatory profile in bronchoalveolar lavage fluid (BALF). The lungs were removed to evaluate alveolar enlargement, extracellular matrix fibers and the expression of MMP-9, MMP-12, TIMP-1, 8-iso-PGF-2α and p65-NF-κB in the fixed tissues as well as to evaluate cytokine levels and p65-NF-κB protein expression.ResultsIn the elastase-treated animals, sakuranetin treatment reduced the alveolar enlargement, collagen and elastic fiber deposition and the number of MMP-9- and MMP-12-positive cells but increased TIMP-1 expression. In addition, sakuranetin treatment decreased the inflammation and the levels of TNF-α, IL-1β and M-CSF in the BALF as well as the levels of NF-κB and 8-iso-PGF-2α in the lungs of the elastase-treated animals. However, this treatment did not affect the changes in lung function.ConclusionThese data emphasize the importance of oxidative stress and metalloproteinase imbalance in the development of emphysema and suggest that sakuranetin is a potent candidate that should be further investigated as an emphysema treatment. This compound may be useful for counteracting lung remodeling and oxidative stress and thus attenuating the development of emphysema.

Electronic supplementary material

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