Systematic classification of melanoma cells by phenotype-specific gene expression mapping

There is growing evidence that the metastatic spread of melanoma is driven not by a linear increase in tumorigenic aggressiveness, but rather by switching back and forth between two different phenotypes of metastatic potential. In vitro these phenotypes are respectively defined by the characteristics of strong proliferation/weak invasiveness and weak proliferation/strong invasiveness. Melanoma cell phenotype is tightly linked to gene expression. Taking advantage of this, we have developed a gene expression-based tool for predicting phenotype called Heuristic Online Phenotype Prediction. We demonstrate the predictive utility of this tool by comparing phenotype-specific signatures with measurements of characteristics of melanoma phenotype-specific biology in different melanoma cell lines and short-term cultures. We further show that 86% of 536 tested melanoma lines and short-term cultures are significantly associated with the phenotypes we describe. These findings reinforce the concept that a two-state system, as described by the phenotype switching model, underlies melanoma progression.     

Identification by 2-D DIGE of apoplastic proteins regulated by oligogalacturonides in Arabidopsis thaliana

Oligogalacturonides (OGs) are elicitors of plant defence responses released from the homogalacturonan of the plant cell wall during the attack by pathogenic micro-organisms. The signalling pathway mediated by OGs remains poorly understood, and no proteins involved in their signal perception and transduction have yet been identified. In order to shed light into the molecular pathways regulated by OGs, a differential proteomic analysis has been carried out in Arabidopsis. Proteins from the apoplastic compartment were isolated and their expression compared between control and OG-treated seedlings. 2-D gels and difference in gel electrophoresis (DIGE) techniques were used to compare control and treated proteomes in the same gel. The analysis of subcellular proteomes from seedlings allowed the identification of novel and low abundance proteins that otherwise remain masked when total cellular extracts are investigated. The DIGE technique showed to be a powerful tool to overcome the high interexperiment variation of 2-D gels. Differentially expressed apoplastic proteins were identified by MS and included proteins putatively involved in recognition as well as proteins whose PTMs are regulated by OGs. Our findings underscore the importance of cell wall as a source of molecules playing a role in the perception of pathogens and provide candidate proteins involved in the response to OGs.     

Taxonomic and phenotypic characterization of yeasts isolated from worldwide cold rock-associated habitats

Yeast strains isolated from rock samples collected from worldwide cold regions were identified by sequence analysis of the D1/D2 domains of the 26S rDNA gene and the ITS region followed by molecular phylogeny. Over 77 % of yeasts isolates were Basidiomycota. Cryptococcus (orders Filobasidiales and Tremellales) and Rhodotorula (order Cystobasidiales) were the most frequent genera. About 40 % of yeast isolates belonged to undescribed species.     

Synthesis, resolution, stereochemistry, and molecular modeling of (R)- and (S)-2-acetyl-1-(4'-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline AMPAR antagonists

Recently we identified (R,S)-2-acetyl-1-(4'-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (6) as a potent non-competitive AMPA receptor antagonist able to prevent epileptic seizures. We report here the optimized synthesis of compound 6, its resolution by chiral preparative HPLC, and the absolute configuration of (R)-enantiomer established by X-ray diffractometry. The biological tests of the single enantiomers revealed that higher anticonvulsant and antagonistic effects reside in (R)-enantiomer as also suggested by molecular modeling studies.     

Double mechanism for apical tryptophan depletion in polarized human bronchial epithelium

Indoleamine 2,3-dioxygenase is an enzyme that catabolizes tryptophan to kynurenine. We investigated the consequences of IDO induction by IFN-gamma in polarized human bronchial epithelium. IDO mRNA expression was undetectable in resting conditions, but strongly induced by IFN-gamma. We determined the concentration of tryptophan and kynurenine in the extracellular medium, and we found that apical tryptophan concentration was lower than the basolateral in resting cells. IFN-gamma caused a decrease in tryptophan concentration on both sides of the epithelium. Kynurenine was absent in control conditions, but increased in the basolateral medium after IFN-gamma treatment. The asymmetric distribution of tryptophan and kynurenine suggested the presence of a transepithelial amino acid transport. Uptake experiments with radiolabeled amino acids demonstrated the presence of a Na(+)-dependent amino acid transporter with broad specificity that was responsible for the tryptophan/kynurenine transport. We confirmed these data by measuring the short-circuit currents elicited by direct application of tryptophan or kynurenine to the apical surface. The rate of amino acid transport was dependent on the transepithelial potential, and we established that in cystic fibrosis epithelia, in which the transepithelial potential is significantly more negative than in noncystic fibrosis epithelia, amino acid uptake was reduced. This work suggests that human airway epithelial cells maintain low apical tryptophan concentrations by two mechanisms, a removal through a Na(+)-dependent amino acid transporter and an IFN-gamma-inducible degradation by IDO.     

Platelet-activating factor mediates CD40-dependent angiogenesis and endothelial-smooth muscle cell interaction

The aim of the present study was to investigate whether stimulation of CD40 expressed by endothelial or smooth muscle cells triggers the synthesis of platelet-activating factor (PAF), an inflammatory mediator with angiogenic properties, and whether PAF contributes to CD40-induced neoangiogenesis. The results obtained indicate that the interaction of CD40 with soluble CD154 or with CD154 expressed on the membrane of leukocytes (CD154-transfected J558 cells) or of activated platelets, stimulated the synthesis of PAF by endothelial cells but not by smooth cells. The synthesis of PAF triggered by activated platelets was inhibited by a soluble CD40-murine Ig fusion protein that prevents the interaction between membrane CD40 and CD154. Studies with specific inhibitors and evaluation of protein phosphorylation indicated the involvement in PAF synthesis of two intracellular signaling pathways leading to cytosolic phospholipase A(2) activation: a phospholipase Cgamma-protein kinase C-Raf-p42/p44-mitogen-activated protein kinase (MAPK) and a MAPK kinase-3/6-dependent activation of p38 MAPK. PAF synthesized by endothelial cells after CD40 stimulation was instrumental in the in vitro migration and vessel-like organization of endothelial cells, and in the interaction between endothelial cells and smooth muscle cells, as inferred by the inhibitory effect of two different PAF receptor antagonists, WEB2170 and CV3988. In vivo, blockade of PAF receptors prevented the angiogenic effect triggered by CD40 stimulation in a murine model of s.c. Matrigel implantation. In conclusion, these observations indicate that PAF synthesis induced by stimulation of endothelial CD40 contributes to the formation and organization of new vessels. This may be relevant in the vascular remodeling associated with tumor and inflammatory neoangiogenesis.     

Soluble Products of Escherichia coli Induce Mitochondrial Dysfunction-Related Sperm Membrane Lipid Peroxidation Which Is Prevented by Lactobacilli

Unidentified soluble factors secreted by E. coli, a frequently isolated microorganism in genitourinary infections, have been reported to inhibit mitochondrial membrane potential (ΔΨm), motility and vitality of human spermatozoa. Here we explore the mechanisms involved in the adverse impact of E. coli on sperm motility, focusing mainly on sperm mitochondrial function and possible membrane damage induced by mitochondrial-generated reactive oxygen species (ROS). Furthermore, as lactobacilli, which dominate the vaginal ecosystem of healthy women, have been shown to exert anti-oxidant protective effects on spermatozoa, we also evaluated whether soluble products from these microorganisms could protect spermatozoa against the effects of E. coli. We assessed motility (by computer-aided semen analysis), ΔΨm (with JC-1 dye by flow cytometry), mitochondrial ROS generation (with MitoSOX red dye by flow cytometry) and membrane lipid-peroxidation (with the fluorophore BODIPY C₁₁ by flow cytometry) of sperm suspensions exposed to E. coli in the presence and in the absence of a combination of 3 selected strains of lactobacilli (L. brevis, L. salivarius, L. plantarum). A Transwell system was used to avoid direct contact between spermatozoa and microorganisms. Soluble products of E. coli induced ΔΨm loss, mitochondrial generation of ROS and membrane lipid-peroxidation, resulting in motility loss. Soluble factors of lactobacilli prevented membrane lipid-peroxidation of E. coli-exposed spermatozoa, thus preserving their motility. In conclusion, sperm motility loss by soluble products of E. coli reflects a mitochondrial dysfunction-related membrane lipid-peroxidation. Lactobacilli could protect spermatozoa in the presence of vaginal disorders, by preventing ROS-induced membrane damage.     

Inhibition of prostate growth and inflammation by the vitamin D receptor agonist BXL-628 (elocalcitol)

The prostate is a target organ of vitamin D receptor (VDR) agonists and represents an extra-renal site of 1,25-dihydroxyvitamin D₃ synthesis, but its capacity to respond to VDR agonists has, so far, been almost exclusively probed for the treatment of prostate cancer. We have analyzed the capacity of VDR agonists to treat benign prostatic hyperplasia (BPH), a complex syndrome characterized by a static component related to prostate overgrowth, a dynamic one responsible for urinary irritative symptoms, and an inflammatory component. Preclinical data demonstrate that VDR agonists, and notably BXL-628 (elocalcitol), reduce the static component of BPH by inhibiting the activity of intra-prostatic growth factors downstream of the androgen receptor, and the dynamic component by targeting bladder cells. In addition, BXL-628 inhibits production of proinflammatory cytokines and chemokines by human BPH cells. These data have led to a proof-of-concept clinical study that has successfully shown arrest of prostate growth in BPH patients treated with BXL-628, with excellent safety. We have documented the anti-inflammatory effects of BXL-628 also in animal models of autoimmune prostatitis, observing a significant reduction of intra-prostatic cell infiltrate following administration of this VDR agonist, at normocalcemic doses, in mice with already established disease. These data extend the potential use of VDR agonists to novel indications that represent important unmet medical needs, and provide a sound rationale for further clinical testing.