Hypoxia enhances proliferation and stemness of human adipose-derived mesenchymal stem cells

The aim of the study was to obtain the highest number of multipotent adipose-derived mesenchymal stem cells (ADMSCs) by using culture conditions which favour cell expansion without loss of mesenchymal stem cells (MSC)-like properties. Based on the assumption that stem cells reside in niches characterized by hypoxic condition, we investigated if the low oxygen tension may improve the proliferation and stemness of ADMSCs. Intact adipose tissue was resected from eight subjects, and the stromal vascular fraction was obtained by using type II collagenase. The heterogeneity of cellular lineages was confirmed by immunophenotypic analysis that showed the presence of leukocytes (CD45+), endothelial cells (CD34+), and pericytes (CD140+). The immunophenotype of confluent ADMSCs was similar to that of bone marrow-derived MSCs, except for the expression of CD34, which was variable (donor-dependent) and inversely correlated to the CD36 expression. ADMSCs showed a high clonal efficiency (94.5 ± 1 %) and were able to generate osteoblastic, chondrocytic and adipocytic lineages. ADMSCs were cultured under normoxic (21 % O₂) and hypoxic (1 % O₂) conditions, and we found that hypoxia significantly favoured ADMSC proliferation and preserved the expression of stemness genes, i.e. Nanog and Sox2. Since hypoxia reflects the microenvironment in which ADMSCs must proliferate and differentiate, the culture in hypoxic condition allows to better understand the biology of these cells and their regenerative potential. Low oxygen concentrations promote cell proliferation and stemness, thus enriching the pool of cells potentially able to differentiate into multi-lineages, and extending the possibility of a long-term expansion.     

Transfected Poly(I:C) Activates Different dsRNA Receptors,Leading to Apoptosis or Immunoadjuvant Response in Androgen-independent Prostate Cancer Cells

Despite the effectiveness of surgery or radiation therapy for the treatment of early-stage prostate cancer (PCa), there is currently no effective strategy for late-stage disease. New therapeutic targets are emerging; in particular, dsRNA receptors Toll-like receptor 3 (TLR3) and cytosolic helicases expressed by cancer cells, once activated, exert a pro-apoptotic effect in different tumors. We previously demonstrated that the synthetic analog of dsRNA poly(I:C) induces apoptosis in the androgen-dependent PCa cell line LNCaP in a TLR3-dependent fashion, whereas only a weak apoptotic effect is observed in the more aggressive and androgen-independent PCa cells PC3 and DU145. In this paper, we characterize the receptors and the signaling pathways involved in the remarkable apoptosis induced by poly(I:C) transfected by Lipofectamine (in-poly(I:C)) compared with the 12-fold higher free poly(I:C) concentration in PC3 and DU145 cells. By using genetic inhibition of different poly(I:C) receptors, we demonstrate the crucial role of TLR3 and Src in in-poly(I:C)-induced apoptosis. Therefore, we show that the increased in-poly(I:C) apoptotic efficacy is due to a higher binding of endosomal TLR3. On the other hand, we show that in-poly(I:C) binding to cytosolic receptors MDA5 and RIG-I triggers IRF3-mediated signaling, leading uniquely to the up-regulation of IFN-β, which likely in turn induces increased TLR3, MDA5, and RIG-I proteins. In summary, in-poly(I:C) activates two distinct antitumor pathways in PC3 and DU145 cells: one mediated by the TLR3/Src/STAT1 axis, leading to apoptosis, and the other one mediated by MDA5/RIG-I/IRF3, leading to immunoadjuvant IFN-β expression.     

Synthesis and in vitro antimicrobial activities of new (cyano-NNO-azoxy)pyrazole derivatives

The antibacterial and antifungal activity of a series of products, in which the 1,5-dimethyl-4-(cyano-NNO-azoxy)pyrazol-3-yl and 1,3-dimethyl-4-(cyano-NNO-azoxy)pyrazol-5-yl moieties were linked to pyridine, pyrazole, isoxazole, thiophene and the furan ring, were examined. No molecule displayed activity against the Gram-negative bacteria tested. Conversely, some compounds displayed activity against two Staphylococcus aureus strains, including the methicillin resistant strain. All compounds displayed interesting antifungal activity, the most active compound of the series being the thiophene derivative 7a. This compound’s activity against Candidakrusei and Candidaglabrata (MIC = 0.25 and 0.5 μg/mL, respectively), two fungal species resistant to azoles, is noteworthy. The presence of the cyano function appeared essential for activity.     

Sensitivity to cisplatin in primary cell lines derived from human glioma correlates with levels of EGR-1 expression

Background  

Less than 30% of malignant gliomas respond to adjuvant chemotherapy. Here, we have asked whether variations in the constitutive expression of early-growth response factor 1 (EGR-1) predicted acute cytotoxicity and clonogenic cell death in vitro, induced by six different chemotherapics.     

A new alpinumisoflavone derivative from Genista pichisermolliana

The aerial parts of Genista pichisermolliana Valsecchi (Fabaceae), an endemic plant of Sardinia, were extracted in Soxhlet apparatus and purified by several chromatographic methods. The new compound alpinumisoflavone 4′-O-glucopyranoside (6) was isolated together with nineteen flavonoids, p-coumaric methylester and d-pinitol, while no alkaloids were detected. All the chemical structures were elucidated by spectroscopic analysis. Since flavonoids represent the main constituents of this plant, the total flavonoid content was determined according to the Italian Pharmacopoeia IX Ed. method.     

Independent roles of apical membrane antigen 1 and rhoptry neck proteins during host cell invasion by apicomplexa

During invasion, apicomplexan parasites form an intimate circumferential contact with the host cell, the tight junction (TJ), through which they actively glide. The TJ, which links the parasite motor to the host cell cytoskeleton, is thought to be composed of interacting apical membrane antigen 1 (AMA1) and rhoptry neck (RON) proteins. Here we find that, in Plasmodium berghei, while both AMA1 and RON4 are important for merozoite invasion of erythrocytes, only RON4 is required for sporozoite invasion of hepatocytes, indicating that RON4 acts independently of AMA1 in the sporozoite. Further, in the Toxoplasma gondii tachyzoite, AMA1 is dispensable for normal RON4 ring and functional TJ assembly but enhances tachyzoite apposition to the cell and internalization frequency. We propose that while the RON proteins act at the TJ, AMA1 mainly functions on the zoite surface to permit correct attachment to the cell, which may facilitate invasion depending on the zoite-cell combination.     

Activation of proteinase-activated receptor-1 inhibits neurally evoked chloride secretion in the mouse colon in vitro

The proteinase-activated thrombin receptor-1 (PAR-1) belongs to a unique family of G protein-coupled receptors activated by proteolytic cleavage. We studied the effect of PAR-1 activation in the regulation of ion transport in mouse colon in vitro. Expression of PAR-1 in mouse colon was assessed by RT-PCR and immunohistochemistry. To study the role of PAR-1 activation in chloride secretion, mouse colon was mounted in Ussing chambers. Changes in short-circuit current (Isc) were measured in tissues exposed to either thrombin, saline, the PAR-1-activating peptide TFLLR-NH2, or the inactive reverse peptide RLLFT-NH2, before electrical field stimulation (EFS). Experiments were repeated in the presence of either a PAR-1 antagonist or in PAR-1-deficient mice to assess receptor specificity. In addition, studies were conducted in the presence of chloride-free buffer or the muscarinic antagonist atropine to assess chloride dependency and the role of cholinergic neurons in the PAR-1-induced effect. PAR-1 mRNA was expressed in full-thickness specimens and mucosal scrapings of mouse colon. PAR-1 immunoreactivity was found on epithelial cells and on neurons in submucosal ganglia where it was colocalized with both VIP and neuropeptide Y. After PAR-1 activation by thrombin or TFLLR-NH2, secretory responses to EFS but not those to forskolin or carbachol were significantly reduced. The reduction in the response to EFS was not observed in the presence of the PAR-1 antagonist, in PAR-1-deficient mice, when chloride was excluded from the bathing medium, or when atropine was present. PAR-1 is expressed in submucosal ganglia in the mouse colon and its activation leads to a decrease in neurally evoked epithelial chloride secretion.     

Glycine residues appear to be evolutionarily conserved for their ability to inhibit aggregation

Six glycine residues of human muscle acylphosphatase (AcP) are evolutionarily conserved across the three domains of life. We have generated six variants of AcP, each having a glycine substituted by an alanine (G15A, G19A, G37A, G45A, G53A, and G69A). Three additional variants had Gly45 replaced by serine, glutamate, and arginine, respectively. The mutational variants do not, on average, have a lower conformational stability than other variants with substitutions of nonconserved residues. In addition, only the G15A variant is enzymatically inactive. However, all variants, with the exception of the G15A mutant, form amyloid aggregates more rapidly than the wild-type. Dynamic light-scattering experiments carried out under conditions close to physiological confirm that aggregate formation is generally more pronounced for the glycine-substituted variants. Apart from the glycine at position 15, all other conserved glycine residues in this protein could have been maintained during evolution because of their ability to inhibit aggregation.     

Involvement of PI3K and MAPK signaling in bcl-2-induced vascular endothelial growth factor expression in melanoma cells

We have previously demonstrated that bcl-2 overexpression in tumor cells exposed to hypoxia increases the expression of vascular endothelial growth factor (VEGF) gene through the hypoxia-inducible factor-1 (HIF-1). In this article, we demonstrate that exposure of bcl-2 overexpressing melanoma cells to hypoxia induced phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2 proteins. On the contrary, no modulation of these pathways by bcl-2 was observed under normoxic conditions. When HIF-1alpha expression was reduced by RNA interference, AKT and ERK1/2 phosphorylation were still induced by bcl-2. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways reduced the induction of VEGF and HIF-1 in response to bcl-2 overexpression in hypoxia. No differences were observed between control and bcl-2-overexpressing cells in normoxia, in terms of VEGF protein secretion and in response to PI3K and MAPK inhibitors. We also demonstrated that RNA interference-mediated down-regulation of bcl-2 expression resulted in a decrease in the ERK1/2 phosphorylation and VEGF secretion only in bcl-2-overexpressing cell exposed to hypoxia but not in control cells. In conclusion, our results indicate, for the first time, that bcl-2 synergizes with hypoxia to promote expression of angiogenesis factors in melanoma cells through both PI3K- and MAPK-dependent pathways.     

Apples increase nitric oxide production by human saliva at the acidic pH of the stomach: a new biological function for polyphenols with a catechol group?

Dietary inorganic nitrate is secreted in saliva and reduced to nitrite by bacterial flora. At the acidic pH of the stomach nitrite is present as nitrous acid in equilibrium with nitric oxide (*NO), and other nitrogen oxides with nitrating and nitrosating activity. *NO in the stomach exerts several beneficial effects, but nitrosating/nitrating species have been implicated as a possible cause of epithelial neoplasia at the gastroesophageal junction. We investigated the effects of apple extracts on *NO release by human saliva at pH 2. A water extract obtained from apple homogenate increased *NO release caused by acidification of saliva. Data show that polyphenols were responsible for this activity, with chlorogenic acid and (+)-catechin the most active and concentrated species. However, ferulic acid, a hydroxycinnamic acid with only one aromatic hydroxyl group, did not increase *NO release. Fructose, the most representative sugar in apples, was also inactive. Interestingly, ascorbic acid in saliva induced a SCN(-)-enhanced burst of *NO but, unlike apple, the release was transient. The simultaneous addition of ascorbic acid and apple extract caused a burst of *NO followed by the increased steady-state level characteristic of saliva containing apple extract. Chlorogenic acid and (+)-catechin, but not ferulic acid, formed o-semiquinone radicals and nitrated polyphenols, suggesting the scavenging of *NO(2) by o-semiquinones. Our results propose that some apple polyphenols not only inhibit nitrosation/nitration but also promote *NO bio-availabilty at the gastric level, a previously unappreciated function.