Protective effect of new S-acylglutathione derivatives against amyloid-induced oxidative stress

Recent data support the role of oxidative stress in the pathogenesis of Alzheimer disease (AD). In particular, glutathione (GSH) metabolism is altered and its levels are decreased in affected brain regions and peripheral cells from AD patients and in experimental models of AD. In the past decade, interest in the protective effects of various antioxidants aimed at increasing intracellular GSH content has been growing. Because much experimental evidence suggests a possible protective role of unsaturated fatty acids in age-related diseases, we designed the synthesis of new S-acylglutathione (acyl-SG) thioesters. S-Lauroylglutathione (lauroyl-SG) and S-palmitoleoylglutathione (palmitoleoyl-SG) were easily internalized into the cells and they significantly reduced Abeta42-induced oxidative stress in human neurotypic SH-SY5Y cells. In particular, acyl-SG thioesters can prevent the impairment of intracellular ROS scavengers, intracellular ROS accumulation, lipid peroxidation, and apoptotic pathway activation. Palmitoleoyl-SG seemed more effective in cellular protection against Abeta-induced oxidative damage than lauroyl-SG, suggesting a valuable role for the monounsaturated fatty acid. In this study, we demonstrate that acyl-SG derivatives completely avoid the sharp lipoperoxidation in primary fibroblasts from familial AD patients occurring after exposure to Abeta42 aggregates. Hence, we put forward these derivatives as new antioxidant compounds which could be excellent candidates for therapeutic treatment of AD and other oxidative stress-related diseases.     

Epithelial-mesenchymal transition of ovarian tumor cells induces an angiogenic monocyte cell population

Vasculogenesis, or recruitment of progenitors able to differentiate into endothelial-like cells, may provide an important contribution to neovessel formation in tumors. However, the factors involved in the vasculogenic process and in particular the role of the epithelial-mesenchymal transition of tumor cells have not yet been investigated. We found a CD14⁺/KDR⁺ angiogenic monocyte population in undifferentiated ovarian tumors, significantly increased in the corresponding tumor metastasis. In vitro, monocyte differentiation into CD14⁺/KDR⁺ cells was induced by conditioned media from the primary ovarian tumor cells expressing a mesenchymal phenotype. In contrast, the ovarian tumor cell line SKOV3 expressing an epithelial phenotype was unable to stimulate the differentiation of monocytes into CD14⁺/KDR⁺ cells. When an epithelial-mesenchymal transition was induced in SKOV3, they acquired this differentiative ability. Moreover, after mesenchymal transition pleiotrophin expression by SKOV3 was increased and conversely its blockade significantly reduced monocyte differentiation. The obtained CD14⁺/KDR⁺ cell population showed the expression of endothelial markers, increased the formation of capillary-like structures by endothelial cells and promoted the migration of ovarian tumor cells in vitro. In conclusion, we showed that the epithelial-mesenchymal transition of ovarian tumor cells induced differentiation of monocytes into the pro-angiogenic CD14⁺/KDR⁺ population and thus it may provide a tumor microenvironment that favours vasculogenesis and metastatization of the ovarian cancer.     

A proteomic approach to paclitaxel chemoresistance in ovarian cancer cell lines

Ovarian cancer is the leading cause of gynaecological cancer mortality. Paclitaxel is used in the first line treatment of ovarian cancer, but acquired resistance represents the most important clinical problem and a major obstacle to a successful therapy. Several mechanisms have been implicated in paclitaxel resistance, however this process has not yet been fully explained. To better understand molecular resistance mechanisms, a comparative proteomic approach was undertaken on the human epithelial ovarian cancer cell lines A2780 (paclitaxel sensitive), A2780TC1 and OVCAR3 (acquired and inherently resistant). Proteins associated with chemoresistance process were identified by DIGE coupled with mass spectrometry (MALDI-TOF and LC-MS/MS). Out of the 172 differentially expressed proteins in pairwise comparisons among the three cell lines, 151 were identified and grouped into ten main functional classes. Most of the proteins were related to the category of stress response (24%), metabolism (22%), protein biosynthesis (15%) and cell cycle and apoptosis (11%), suggesting that alterations of those processes might be involved in paclitaxel resistance mechanisms. This is the first direct proteomic comparison of paclitaxel sensitive and resistant ovarian cancer cells and may be useful for further studies of resistance mechanisms and screening of resistance biomarkers for the development of tailored therapeutic strategies.     

Microparticles Carrying Sonic Hedgehog Favor Neovascularization through the Activation of Nitric Oxide Pathway in Mice

Background

Microparticles (MPs) are vesicles released from plasma membrane upon cell activation and during apoptosis. Human T lymphocytes undergoing activation and apoptosis generate MPs bearing morphogen Shh (MPs^Shh+) that are able to regulate in vitro angiogenesis.

Methodology/Principal Findings

Here, we investigated the ability of MPs^Shh+ to modulate neovascularization in a model of mouse hind limb ischemia. Mice were treated in vivo for 21 days with vehicle, MPs^Shh+, MPs^Shh+ plus cyclopamine or cyclopamine alone, an inhibitor of Shh signalling. Laser doppler analysis revealed that the recovery of the blood flow was 1.4 fold higher in MPs^Shh+-treated mice than in controls, and this was associated with an activation of Shh pathway in muscles and an increase in NO production in both aorta and muscles. MPs^Shh+-mediated effects on flow recovery and NO production were completely prevented when Shh signalling was inhibited by cyclopamine. In aorta, MPs^Shh+ increased activation of eNOS/Akt pathway, and VEGF expression, being inhibited by cyclopamine. By contrast, in muscles, MPs^Shh+ enhanced eNOS expression and phosphorylation and decreased caveolin-1 expression, but cyclopamine prevented only the effects of MPs^Shh+ on eNOS pathway. Quantitative RT-PCR revealed that MPs^Shh+ treatment increased FGF5, FGF2, VEGF A and C mRNA levels and decreased those of α5-integrin, FLT-4, HGF, IGF-1, KDR, MCP-1, MT1-MMP, MMP-2, TGFβ1, TGFβ2, TSP-1 and VCAM-1, in ischemic muscles.

Conclusions/Significance

These findings suggest that MPs^Shh+ may contribute to reparative neovascularization after ischemic injury by regulating NO pathway and genes involved in angiogenesis.     

Olive embryo in vitro germination potential: role of explant configuration and embryo structure among cultivars

The in vitro germination of excised embryos can break dormancy rapidly and shorten the time required to produce seedlings, speeding up olive breeding programmes as well as rootstock production. In this study, the in vitro germination potential of four Sicilian olive cultivars was evaluated during two years of experiments, using explants with three different morphological configurations that represent three different degrees of embryo exposure: (1) intact stoneless seeds containing the embryo, the endosperm and the seed coat (Emb+En+SC), (2) seeds without the seed coat (Emb+En) and (3) naked, isolated embryos (seed coat and endosperm both removed: Emb). Differences were found in the germination percentages and timing due to both genotype and explant type. The root and shoot meristems, the radicle-hypocotyl axis, the provascular tissues and embryo storage reserves were identified as embryo anatomical structures which could influence germination capacity. Observation of these structures, however, indicated similar germination potential among cultivars, suggesting possible differences in other dormancy factors. In spite of variation in cultivar performance, after 60 days of in vitro culture all cultivars demonstrated the highest germination of naked embryos (explant type 3) and lowest for stoneless seeds (explant type 1); stoneless seeds without the seedcoat (explant type 2) showed intermediate germination percentages.     

Reverse engineering of protein secretion by uncoupling of cell cycle phases from growth

The demand for recombinant proteins both for biopharmaceutical and technical applications is rapidly growing, and therefore the need to establish highly productive expression systems is steadily increasing. Yeasts, such as Pichia pastoris, are among the widely used production platforms with a strong emphasis on secreted proteins. Protein secretion is a limiting factor of productivity. There is strong evidence that secretion is coupled to specific growth rate (µ) in yeast, being higher at higher µ. For maximum productivity and product titer, high specific secretion rates at low µ would be desired. At high secretion rates cultures contain a large fraction of cells in the G2 and M phases of cell cycle. Consequently, the cell design target of a high fraction of cells in G2 + M phase was achieved by constitutive overexpression of the cyclin gene CLB2. Together with predictive process modeling this reverse engineered production strain improved the space time yield (STY) of an antibody Fab fragment by 18% and the product titer by 53%. This concept was verified with another secreted protein, human trypsinogen. Biotechnol. Bioeng. 2011;108: 2403–2412. © 2011 Wiley Periodicals, Inc.     

Production of recombinant proteins and metabolites in yeasts

Recombinant DNA (rDNA) technologies allow the production of a wide range of peptides, proteins and metabolites from naturally non-producing cells. Since human insulin was the first heterologous compound produced in a laboratory in 1977, rDNA technology has become one of the most important technologies developed in the 20th century. Recombinant protein and metabolites production is a multi-billion dollar market. The development of a new product begins with the choice of the cell factory. The final application of the compound dictates the main criteria that should be taken into consideration: (1) quality, (2) quantity, (3) yield and (4) space time yield of the desired product. Quantity and quality are the most predominant requirements that must be considered for the commercial production of a protein. Quantity and yield are the requirements for the production of a metabolite. Finally, space time yield is crucial for any production process. It therefore becomes clear why the perfect host does not exist yet, and why—despite important advances in rDNA applications in higher eukaryotic cells—microbial biodiversity continues to represent a potential source of attractive cell factories. In this review, we compare the advantages and limitations of the principal yeast and bacterial workhorse systems.