Synthesis, spectroscopic, structural and electrochemical studies of carboxyl substituted 1,4-naphthoquinones

Two carboxyl substituted quinones and their ethyl esters were prepared by alkylation of 2-methyl-1,4-naphthoquinone (MNQ), also known as menadione or vitamin K₃. All products were characterized by spectroscopic (¹H NMR, ¹³C NMR, IR) and electrochemical (cyclic voltammetry) methods, and the crystal structure of the two carboxylic derivatives was also determined. Both carboxyl substituted quinones crystallize in the system as hydrogen bonded dimers. In MeCN, the cyclic voltammograms of the ester derivatives present two reversible one-electron redox waves very similar to those of the parent quinone, MNQ. However, in the same solvent, the corresponding carboxyl substituted quinones show one cathodic and one anodic additional irreversible waves at more positive potentials and a decrease in current intensity of the two quinone reduction waves accompanied by loss of the quasi-reversible character of the second wave. These results show that the presence of the carboxylic substituent does not greatly modify the redox behaviour of the quinone, except for a small anodic shift of the potentials, but the associated presence of H⁺ ions in solution causes an important perturbation to the system, stabilizing the electrogenerated semiquinones by intermolecular self-protonation and/or hydrogen bonding.     

Modelling potential habitat of the invasive ctenophore <Emphasis Type="Italic">Mnemiopsis leidyi</Emphasis> in Aegean Sea

The invasive ctenophore Mnemiopsis leidyi was accidentally introduced into the Black Sea in the early 1980s and it was first sighted in the Aegean Sea (Eastern Mediterranean) in the early 1990s. This article presents a first attempt to develop a predictive spatial model based on M. leidyi presence data and satellite environmental data from the Aegean Sea during early summer, in order to identify those areas in the Greek Seas and the entire Mediterranean basin that could serve as potential habitat for the species. Generalized additive models (GAM) were applied. The final GAM model indicated higher probability of finding M. leidyi present in depths of 65–135 m and sea surface temperature values of 21–25°C. Furthermore, the significant interaction between photosynthetically active radiation (PAR) and sea level anomaly (SLA) indicated a higher probability of M. leidyi presence in low values of PAR and SLA. In the next step, the final GAM was applied in a prediction grid of mean monthly satellite values for June 2004–2006 in order to estimate probability of M. leidyi presence in the Hellenic Seas and the whole Mediterranean basin at a GIS resolution of 4 km. In the Aegean Sea, species potential habitat included areas influenced by the Black Sea Water (e.g. Thracian Sea, Limnos-Imvros plateau), gulfs that are affected by river runoffs, such as the Thermaikos, Strymonikos and Patraikos gulfs, or areas with strong anthropogenic influence such as the Saronikos gulf. Areas with the same environmental conditions as those in Aegean Sea have been indicated in certain spots of the Levantine Sea as well as in coastal waters of Egypt and Libya, although their spatial extent varied largely among years examined. However, the occurrence of conditions that are linked to high probability of M. leidyi presence does not necessarily mean that these areas can support successful reproduction, high population or bloom levels, since these depend on a combination of temperature, salinity, food availability and the abundance of predators. Guest editor: V. D. Valavanis Essential Fish Habitat Mapping in the Mediterranean     

Development and characterization of a tissue engineered pancreatic substitute based on recombinant intestinal endocrine L‐cells

A tissue engineered pancreatic substitute (TEPS) consisting of insulin‐producing cells appropriately designed and encapsulated to support cellular function and prevent interaction with the host may provide physiological blood glucose regulation for the treatment of insulin dependent diabetes (IDD). The performance of agarose‐based constructs which contained either a single cell suspension of GLUTag‐INS cells, a suspension of pre‐aggregated GLUTag‐INS spheroids, or GLUTag‐INS cells on small intestinal submucosa (SIS), was evaluated in vitro for total cell number, weekly glucose consumption and insulin secretion rates (GCR and ISR), and induced insulin secretion function. The three types of TEPS studied displayed similar number of cells, GCR, and ISR throughout 4 weeks of culture. However, the TEPS, which incorporated SIS as a substrate for the GLUTag‐INS cells, was the only type of TEPS tested which was able to retain the induced insulin secretion function of non‐encapsulated GLUTag‐INS cells. Though improvements in the expression level of GLUTag‐INS cells and/or the number of viable cells contained within the TEPS are needed for successful treatment of a murine model of IDD, this study has revealed a potential method for promoting proper cellular function of recombinant L‐cells upon incorporation into an implantable three‐dimensional TEPS. Biotechnol. Bioeng. 2009;103: 828–834. © 2009 Wiley Periodicals, Inc.     

The Mechanism of CSF-1-induced Wiskott-Aldrich Syndrome Protein Activation in Vivo: A ROLE FOR PHOSPHATIDYLINOSITOL 3-KINASE AND Cdc42*

A role for Wiskott-Aldrich syndrome protein (WASP) in chemotaxis to various agents has been demonstrated in monocyte-derived cell types. Although WASP has been shown to be activated by multiple mechanisms in vitro, it is unclear how WASP is regulated in vivo. A WASP biosensor (WASPbs), which uses intramolecular fluorescence resonance energy transfer to report WASP activation in vivo, was constructed, and following transfection of macrophages, activation of WASPbs upon treatment with colony-stimulating factor-1 (CSF-1) was detected globally as early as 30 s and remained localized to protrusive regions at later time points. Similar results were obtained when endogenous WASP activation was determined using conformation-sensitive antibodies. In vivo CSF-1-induced WASP activation was fully Cdc42-dependent. Activation of WASP in response to treatment with CSF-1 was also shown to be phosphatidylinositol 3-kinase-dependent. However, treatment with the Src family kinase inhibitors PP2 or SU6656 or disruption of the major tyrosine phosphorylation site of WASPbs (Y291F mutation) did not reduce the level of CSF-1-induced WASP activation. Our results indicate that WASP activation downstream of CSF-1R is phosphatidylinositol 3-kinase- and Cdc42-dependent consistent with an involvement of these molecules in macrophage migration. However, although tyrosine phosphorylation of WASP has been proposed to stimulate WASP activity, we found no evidence to indicate that this occurs in vivo.Macrophages, terminally differentiated cells of the mononuclear phagocytic lineage, are found throughout the body and play important roles in normal tissue development and immune defense. However, in certain circumstances, excessive recruitment of macrophages has been shown to participate in the progression of several diseases, inflammatory (rheumatoid arthritis) or metabolic (atherosclerosis), as well as in tumor progression (1–3). Importantly expression of colony-stimulating factor-1 (CSF-1),⁴ the most pleiotropic macrophage growth factor, has been correlated with the progression of these disease states (for a review, see Ref. 4). Inhibition of undesirable macrophage recruitment to specific sites in response to CSF-1 is therefore an attractive goal for therapies (5).In addition to stimulating survival, proliferation, and differentiation of monocytes and macrophages, CSF-1 is also a potent chemotactic factor inducing the migration of these cell types (for a review, see Ref. 4). CSF-1 stimulation leads to the rapid production of F-actin-rich protrusions and the spreading and migration of macrophages (4). All CSF-1 effects are mediated through its tyrosine kinase receptor (CSF-1R), which upon activation leads to phosphorylation of tyrosine residues in a number of signaling molecules. Downstream molecules essential for macrophage migration in response to CSF-1 include phosphatidylinositol 3-kinase (PI3K) isoforms β and δ (6, 7). PI3K may potentially regulate migration through the activation of guanine nucleotide exchange factor activity to Rac1 and Cdc42, which are required for CSF-1-elicited protrusions (8, 9) and chemotaxis (10). The major means by which Rac and Cdc42 regulate the Arp2/3 complex is through the Wiskott-Aldrich syndrome protein/Wiskott-Aldrich syndrome verprolin-homologous (WASP/WAVE) family of proteins (11). A Rac1-IRSp53-Abi1-WAVE2 complex has been shown to mediate CSF-1-induced macrophage motility (12, 13), and a unique role for WASP in macrophage chemotaxis to CSF-1, formylmethionylleucylphenylalanine, MCP-1, and MIP-1α has been demonstrated (14, 15). WASP is a hematopoietic cell-specific regulator of Arp2/3-dependent actin remodeling. The catalytically active domain of WASP lies in its C terminus, which is conserved among all WASP/WAVE proteins and contains a VCA (verprolin homology, cofilin-like, and acidic region) domain capable of activating the Arp2/3 complex. The other domains found in WASP can regulate, directly or indirectly, the activity of its VCA domain (for a review, see Ref. 16). Both WASP and N-WASP bind activated Cdc42 through their GTPase-binding domain, which is believed to cause a structural transition that results in dissociation of the intramolecular contacts leaving the VCA domain accessible for Arp2/3 binding (17, 18). In addition, biochemical studies have revealed that several signaling molecules, including WASP-interacting SH3 protein, WASP-interacting protein, Grb2, phosphoinositides, and Src family kinases, activate N-WASP (for reviews, see Refs. 16 and 19). Phosphorylation of WASP has also been proposed to activate Arp2/3-mediated actin polymerization in vitro (20–22).Recently different probes have been developed that detect a conformational change in N-WASP and therefore reflect its activation (23–25). Using either a fluorescence resonance energy transfer (FRET)-based biosensor that detects a conformational change in N-WASP (23, 24) or antibodies that can only bind to the open conformation of N-WASP (25), N-WASP has been shown to be activated in response to epidermal growth factor in HEK293 cells and in MTLn3 carcinoma cells. This activity has been temporally localized to subcellular compartments important for carcinoma cell chemotaxis and invasion (24). We have adapted these approaches to explore the signal transduction pathways responsible for the activation of WASP in vivo.     

Sirtuins at the crossroads of stemness,aging, and cancer

Sirtuins are stress‐responsive proteins that direct various post‐translational modifications (PTMs) and as a result, are considered to be master regulators of several cellular processes. They are known to both extend lifespan and regulate spontaneous tumor development. As both aging and cancer are associated with altered stem cell function, the possibility that the involvement of sirtuins in these events is mediated by their roles in stem cells is worthy of investigation. Research to date suggests that the individual sirtuin family members can differentially regulate embryonic, hematopoietic as well as other adult stem cells in a tissue‐ and cell type‐specific context. Sirtuin‐driven regulation of both cell differentiation and signaling pathways previously involved in stem cell maintenance has been described where downstream effectors involved determine the biological outcome. Similarly, diverse roles have been reported in cancer stem cells (CSCs), depending on the tissue of origin. This review highlights the current knowledge which places sirtuins at the intersection of stem cells, aging, and cancer. By outlining the plethora of stem cell‐related roles for individual sirtuins in various contexts, our purpose was to provide an indication of their significance in relation to cancer and aging, as well as to generate a clearer picture of their therapeutic potential. Finally, we propose future directions which will contribute to the better understanding of sirtuins, thereby further unraveling the full repertoire of sirtuin functions in both normal stem cells and CSCs.     

MKK7 and ARF

Sensing, integrating, and processing of stressogenic signals must be followed by accurate differential response(s) for a cell to survive and avoid malignant transformation. The DNA damage response (DDR) pathway is vital in this process, as it deals with genotoxic/oncogenic insults, having p53 as a nodal effector that performs most of the above tasks. Accumulating data reveal that other pathways are also involved in the same or similar processes, conveying also to p53. Emerging questions are if, how, and when these additional pathways communicate with the DDR axis. Two such stress response pathways, involving the MKK7 stress-activated protein kinase (SAPK) and ARF, have been shown to be interlocked with the ATM/ATR-regulated DDR axis in a highly ordered manner. This creates a new landscape in the DDR orchestrated response to genotoxic/oncogenic insults that is currently discussed.     

Molecular Characterization of Recombinant Mus a 5 Allergen from Banana Fruit

Allergy to banana fruit appears to have become an important cause of fruit allergy in Europe. Among five allergens that have been found, beta-1,3-glucanase denoted as Mus a 5 was identified as a candidate allergen for the component-resolved allergy diagnosis of banana allergy. Because of the variations in protein levels in banana fruit, in this study Mus a 5 was produced as a fusion protein with glutathione-S-transferase in Escherichia coli. The recombinant Mus a 5 was purified under native conditions by a combination of affinity, ion-exchange, and reversed phase chromatography. N-terminal sequence was confirmed by Edman degradation and 55 % of the primary structure was identified by mass fingerprint, while the secondary structure was assessed by circular dichroism spectroscopy. IgG reactivity of recombinant protein was shown in 2-D immunoblot with anti-Mus a 5 antibodies, while IgG and IgE binding to natural Mus a 5 was inhibited with the recombinant Mus a 5 in immunoblot inhibition test. IgE reactivity of recombinant Mus a 5 was shown in ELISA within a group of ten persons sensitized to banana fruit. Recombinant Mus a 5 is a novel reagent suitable for the component-resolved allergy diagnosis of banana allergy.     

Crosstalk between the DNA damage response,histone modifications and neovascularisation

Neovascularisation is critical in several malignant and inflammatory conditions, as well as in the course of eye disorders. During new vessel formation, endothelial cell functions, such as proliferation and sprouting are very important and are regulated by a variety of growth factors. The DNA damage response machinery as well as factors regulating histone modifications, such as histone deacetylases, regulate cell fate as well as gene expression. Recent evidence has pointed to potential interactions among BRCA1, H2AX and SIRT1 in these intracellular pathways and neovascularisation, which will be reviewed here.     

Cyclic AMP and Butyrate Modulate Melatonin Synthesis in Y79 Human Retinoblastoma Cells

Abstract: Melatonin is synthesized by cultured Y79 human retinoblastoma cells and is secreted into the medium. Activity of the two key enzymes involved in the synthesis of melatonin, N -acetyltransferase (NAT) and hydroxyindole- O -methyl-transferase (HIOMT), are present in retinoblastoma cells. The activity of these enzymes and the resulting synthesis and release of melatonin are modulated by the addition of a cyclic AMP analogue and butyrate to the culture medium. Melatonin levels increase dramatically over control levels after the addition of dibutyryl cyclic AMP (dbcAMP), whereas melatonin levels decrease after butyrate treatment. HIOMT activity is inhibited by both dbcAMP and butyrate, and NAT activity is stimulated by both of these differentiating agents, suggesting that the rise in melatonin levels in response to dbcAMP is the result of increased activity of NAT, whereas the decline in melatonin levels in response to butyrate may be due to a drop in HIOMT activity. Melatonin synthesis is dose- and time-dependent, and the effect of dbcAMP is readily reversible, whereas the effect of butyrate does not appear to be reversible. These effects probably reflect basic differences in the regulatory mechanisms of the inducing agents.