Effects of Different Heating Treatments on the Antioxidant Activity and Phenolic Compounds of Ecuadorian Red Dacca Banana.

The banana is a tropical fruit characterized by its composition of healthy and nutritional compounds. This fruit is part of traditional Ecuadorian gastronomy, being consumed in a wide variety of ways. In this context, unripe Red Dacca banana samples and those submitted to different traditional Ecuadorian heating treatments (boiling, roasting, and baking) were evaluated to profile their phenolic content by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) and the antioxidant activity by ORAC, ABTS, and DPPH assays. A total of sixty-eight phenolic compounds were identified or tentatively identified in raw banana and treated samples, highlighting the content in flavonoids (flavan-3-ols with 88.33% and flavonols with 3.24%) followed by the hydroxybenzoic acid family (5.44%) in raw banana samples. The total phenolic compound content significantly decreased for all the elaborations evaluated, specifically from 442.12 mg/100 g DW in fresh bananas to 338.60 mg/100 g DW in boiled (23.41%), 243.63 mg/100 g DW in roasted (44.90%), and 109.85 mg/100 g DW in baked samples (75.15%). Flavan-3-ols and flavonols were the phenolic groups most affected by the heating treatments, while flavanones and hydroxybenzoic acids showed higher stability against the heating treatments, especially the boiled and roasted samples. In general, the decrease in phenolic compounds corresponded with a decline in antioxidant activity, evaluated by different methods, especially in baked samples. The results obtained from PCA studies confirmed that the impact of heating on the composition of some phenolic compounds was different depending on the technique used. In general, the heating processes applied to the banana samples induced phytochemical modifications. Even so, they remain an important source of bioactive compounds for consumers.

Adenoviral delivery of soluble ovine OX40L or CD70 costimulatory molecules improves adaptive immune responses to a model antigen in sheep.

The tumour necrosis factor superfamily OX40L and CD70 and their receptors are costimulatory signalling axes critical for adequate T and B cell activation in humans and mice. In this work we inoculated groups of sheep with human recombinant adenovirus type 5 (Ad) expressing Ovis aries (Oa)OX40L or OaCD70 or a control adenoviral vector to determine whether they could improve the immune response to the model antigen OVA. PBMCs and serum samples were obtained for analysis of the adaptive immune response to OVA at days 0, 15, 30 and 90 post-inoculation (pi). Recall responses to OVA were assessed at day 7 and 30 after the second antigen inoculation (pb) at day 90. Administration of these immunomodulatory molecules did not induce unspecific PBMC stimulation. While OaOX40L administration mainly increased TNF-α and IL-4 in PBMC at day 15 pi concomitantly with a slight increase in antibody titer and the number of IFN-γ producing cells, we detected greater effects on adaptive immunity after OaCD70 administration. AdOaCD70 inoculation improved antibody titers to OVA at days 30 and 90 pi, and increased anti-OVA-specific IgG-secreting B cell counts when compared to control. Moreover, higher IFN-γ production was detected on days 7 pi, 7 pb and 30 pb in PBMCs from this group. Phenotypic analysis of T cell activation showed an increase in effector CD8+ T cells (CD8+ CD62L- CD27-) at day 15 pi in AdOaCD70 group, concurrent with a decrease in early activated cells (CD8+ CD62L- CD27+). Moreover, recall anti-OVA CD8+ T cell responses were increased at 7 pb in the AdOaCD70 group. AdOaCD70 administration could therefore promote CD8+ T cell effector differentiation and long-term activity. In this work we characterized the in vivo adjuvant potential on the humoral and cellular immune response of OaOX40L and OaCD70 delivered by non-replicative adenovirus vectors using the model antigen OVA. We present data highlighting the potency of these molecules as veterinary vaccine adjuvant.

A New Look at Vaccine Strategies Against PPRV Focused on Adenoviral Candidates.

Peste des petits ruminants virus (PPRV) is a virus that mainly infects goats and sheep causing significant economic loss in Africa and Asia, but also posing a serious threat to Europe, as recent outbreaks in Georgia (2016) and Bulgaria (2018) have been reported. In order to carry out the eradication of PPRV, an objective set for 2030 by the Office International des Epizooties (OIE) and the Food and Agriculture Organization of the United Nations (FAO), close collaboration between governments, pharmaceutical companies, farmers and researchers, among others, is needed. Today, more than ever, as seen in the response to the SARS-CoV2 pandemic that we are currently experiencing, these goals are feasible. We summarize in this review the current vaccination approaches against PPRV in the field, discussing their advantages and shortfalls, as well as the development and generation of new vaccination strategies, focusing on the potential use of adenovirus as vaccine platform against PPRV and more broadly against other ruminant pathogens.

Magnetic Nanoparticles Attached to the NK Cell Surface for Tumor Targeting in Adoptive Transfer Therapies Does Not Affect Cellular Effector Functions.

Adoptive cell transfer therapy is currently one of the most promising approaches for cancer treatment. This therapy has some limitations, however, such as the dispersion of in vivo-administered cells, causing only a small proportion to reach the tumor. Nanotechnological approaches could offer a solution for this drawback, as they can increase cell retention and accumulation in a region of interest. In particular, strategies employing magnetic nanoparticles (MNPs) to improve targeting of adoptively transferred T or NK cells have been explored in mice. In vivo magnetic retention is reported using the human NK cell line NK-92MI transfected with MNPs. Primary NK cells are nonetheless highly resistant to transfection, and thus we explore in here the possibility of attaching the MNPs to the NK cell surface to overcome this issue, and examine whether this association would affect NK effector functions. We assessed the attachment of MNPs coated with different polymers to the NK cell surface, and found that APS-MNP attached more efficiently to the NK-92MI cell surface. In association with MNPs, these cells preserved their main functions, exhibiting a continued capacity to degranulate, conjugate with and lyse target cells, produce IFN-γ, and respond to chemotactic signals. MNP-loaded NK-92MI cells were also retained in an in vitro capillary flow system by applying an EMF. A similar analysis was carried out in primary NK cells, isolated from mice, and expanded in vitro. These primary murine NK cells also maintained their functionality intact after MNP treatment and were successfully retained in vitro. This work therefore provides further support for using MNPs in combination with EMFs to favor specific retention of functional NK cells in a region of interest, which may prove beneficial to adoptive cell-therapy protocols.

Effect of a grapevine-shoot waste extract on red wine aromatic properties.

BACKGROUND: The use of a grapevine-shoot extract (VIN) is being studied as an alternative to sulfur dioxide (SO2 ). VIN stabilizes anthocyanins and preserves polyphenolic compounds, and thus improves chromatic wine properties. In this study, selected aroma compounds (esters, C13 -norisoprenoids, oxidation and vine-shoot-related compounds), sensory analysis and the olfactometric profile were determined in the wines treated with VIN at two concentrations. RESULTS: Treatment with VIN hardly modified the content of esters and oxidation-related compounds in the wines. However, the high ß-damascenone and isoeugenol contents and the increase in astringency at tasting in VIN wines were noteworthy, as were some odorant zones. All these were established as VIN markers after the chemometric data analysis. CONCLUSION: These data revealed that only the lowest dose tested may be recommended as a suitable alternative to SO2 . Although some aromatic properties of these wines may change, these changes are not considered to affect the quality of the wines negatively. These results are useful for wineries, which face having to discover the aroma-related processes in the challenge of producing SO2 -free wines without detriment to their sensory properties. © 2018 Society of Chemical Industry.

Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders

Background Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.

Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders

Loxoscelism is a severe human envenomation caused by Loxosceles spider venom. To the best of our knowledge, no study has evaluated the presence of antibodies against Loxosceles venom in loxoscelism patients without treatment with antivenom immunotherapy. We perform a comparative analysis for the presence of antibodies capable of recognizing Loxosceles venom in a group of patients diagnosed with loxoscelism and in a group of people without loxoscelism. Methods The detection of L. laeta venom, Sicarius venom and recombinant phospholipases D from Loxosceles (PLDs) in sera from people with loxoscelism (Group 1) and from healthy people with no history of loxoscelism (Group 2) was evaluated using immuno-dot blot, indirect ELISA, and Western blot. Results We found naturally heterophilic antibodies (IgG-type) in people without contact with Loxosceles spiders or any clinical history of loxoscelism. Either serum pools or single sera from Group 1 and Group 2 analyzed by dot blot tested positive for L. laeta venom. Indirect ELISA for venom recognition showed titles of 1:320 for Group 1 sera and 1:160 for Group 2 sera. Total IgG quantification showed no difference in sera from both groups. Pooled sera and purified IgG from sera of both groups revealed venom proteins between 25 and 32 kDa and the recombinant phospholipase D isoform 1 (rLlPLD1), specifically. Moreover, heterophile antibodies cross-react with PLDs from other Loxosceles species and the venom of Sicarius spider. Conclusions People without contact with the spider venom produced heterophilic antibodies capable of generating a cross-reaction against the venom of L. laeta and Sicarius spiders. Their presence and possible interference should be considered in the development of immunoassays for Loxosceles venom detection.(AU)

Time-course assessment of the aggregation and metabolization of magnetic nanoparticles.

To successfully develop biomedical applications for magnetic nanoparticles, it is imperative that these nanoreagents maintain their magnetic properties in vivo and that their by-products are safely metabolized. When placed in biological milieu or internalized into cells, nanoparticle aggregation degree can increase which could affect magnetic properties and metabolization. To evaluate these aggregation effects, we synthesized citric acid-coated iron oxide nanoparticles whose magnetic susceptibility can be modified by aggregation in agar dilutions and dextran-layered counterparts that maintain their magnetic properties unchanged. Macrophage models were used for in vitro uptake and metabolization studies, as these cells control iron homeostasis in the organism. Electron microscopy and magnetic susceptibility studies revealed a cellular mechanism of nanoparticle degradation, in which a small fraction of the particles is rapidly degraded while the remaining ones maintain their size. Both nanoparticle types produced similar iron metabolic profiles but these profiles differed in each macrophage model. Thus, nanoparticles induced iron responses that depended on macrophage programming. In vivo studies showed that nanoparticles susceptible to changes in magnetic properties through aggregation effects had different behavior in lungs, liver and spleen. Liver ferritin levels increased in these animals showing that nanoparticles are degraded and their by-products incorporated into normal metabolic routes. These data show that nanoparticle iron metabolization depends on cell type and highlight the necessity to assess nanoparticle aggregation in complex biological systems to develop effective in vivo biomedical applications. STATEMENT OF SIGNIFICANCE: Magnetic iron oxide nanoparticles have great potential for biomedical applications. It is however imperative that these nanoreagents preserve their magnetic properties once inoculated, and that their degradation products can be eliminated. When placed in a biological milieu nanoparticles can aggregate and this can affect their magnetic properties and their degradation. In this work, we showed that iron oxide nanoparticles trigger the iron metabolism in macrophages, the main cell type involved in iron homeostasis in the organism. We also show that aggregation can affect nanoparticle magnetic properties when inoculated in animal models. This work confirms iron oxide nanoparticle biocompatibility and highlights the necessity to assess in vivo nanoparticle aggregation to successfully develop biomedical applications.

PI3K p85 ß regulatory subunit deficiency does not affect NK cell differentiation and increases NKG2D-mediated activation.

Activation of NK cells depends on a balance between activating and inhibitory signals. Class Ia PI3K are heterodimeric proteins with a catalytic and a regulatory subunit and have a central role in cell signaling by associating with tyrosine kinase receptors to trigger signaling cascades. The regulatory p85 subunit participates in signaling through NKG2D, one of the main activating receptors on NK cells, via its interaction with the adaptor protein DAP10. Although the effects of inhibiting catalytic subunits or deleting the regulatory p85α subunit have been studied, little attention has focused on the role of the p85ß subunit in NK cells. Using p85ß knockout mice, we found that p85ß deficiency does not alter NK cell differentiation and maturation in spleen or bone marrow. NK cells from p85ß-/- mice nonetheless produced more IFN-γ and degranulated more effectively when stimulated with anti-NKG2D antibody. These cells also degranulated and killed NKG2D ligand-expressing target cells more efficiently. We show that p85ß deficiency impaired NKG2D internalization, which could contribute to the activated phenotype. Decreasing p85ß subunit protein levels might thus constitute a therapeutic target to promote NK cell activity toward NKG2D ligand-expressing cells.

Influence of genotype, cultivation system and irrigation regime on antioxidant capacity and selected phenolics of blueberries (Vaccinium corymbosum L.).

Demand for and availability of blueberries has increased substantially over recent years, driven in part by their health-promoting properties. Three blueberry varieties ('Rocío', V2, and V3) were grown under two cultivation systems (open-field and plastic tunnels) and subjected to two irrigations regimes (100% and 80% of crop evapotranspiration) in two consecutive years (2011-2012). They were evaluated for their phytochemical composition and antioxidant capacity. Genotype influenced the antioxidant capacity and the content of the three groups of phenolics in the blueberries. The antioxidant activity and total flavonols content increased when the blueberries were grown under open-field conditions. Deficit irrigation conditions led to additional positive effects on their phenolics (delphinidn-3-acetilhexoside content was increased under plastic tunnel with deficit irrigation). In conclusion, the amount of phenolic compounds and the antioxidant capacity of blueberries were not negatively affected by water restriction; Moreover, several changes were recorded due to growing system and genotype.

Superparamagnetic iron oxide nanoparticle uptake alters M2 macrophage phenotype, iron metabolism, migration and invasion.

Superparamagnetic iron oxide nanoparticles (SPIONs) have shown promise as contrast agents and nanocarriers for drug delivery. Their impact on M2-polarised macrophages has nonetheless not been well studied. Here we explored the effects of SPIONs coated with dimercaptosuccinic acid, aminopropyl silane or aminodextran in two M2 macrophage models (murine primary IL-4-activated bone marrow-derived macrophages and human M2-like differentiated THP-1 cells). All SPIONs were internalised and no cell toxicity was observed. SPION treatment produced reactive oxygen species and activated the extracellular signal-regulated kinase and AKT pathways. After 24-h SPION incubation, M2 macrophages switched their iron metabolism towards an iron-replete state. SPION treatment in both M2 macrophage models altered their M2 activation profiles, promoted IL-10 production, and stimulated protease-dependent invasion. These results highlight the need to evaluate the interactions between SPIONs and cells to take full advantage of the intrinsic properties of these nanoparticles in biological systems. FROM THE CLINICAL EDITOR: Superparamagnetic iron oxide nanoparticles (SPIONs) have been used as an MRI contrast agent in many experimental studies. The authors here investigated the effects of these nanoparticles on M2 macrophages after cellular uptake. The findings of cell activation further enhanced our current knowledge on the interaction of SPIONS with macrophages.

Polyethylenimine-coated SPIONs trigger macrophage activation through TLR-4 signaling and ROS production and modulate podosome dynamics.

Polyethylenimine (PEI) is widely used as transfection agent in preclinical studies, both in vitro and in vivo. Due to their unique chemical and physical properties, SPIONs (superparamagnetic iron oxide nanoparticles) have been thoroughly studied as nanocarriers. PEI appears to activate different immune cells to an inflammatory response (M1/TH1), whereas the SPION-induced response seems to be context-dependent; the immunogenicity of the combination of these components has not been studied. Here we show that PEI-coated SPIONs (PMag) activate macrophages, as determined by measuring IL-12 secretion into culture medium and upregulation of several genes linked to the M1 phenotype. PMag-induced phosphorylation of p38 MAPK, p44/p42 MAPK and JNK, and upregulation of CD40, CD80, CD86 and I-A/I-E activation markers. PMag-induced macrophage activation depended partially on TLR4 (Toll-like receptor 4) and ROS (reactive oxygen species) signaling. Comparison of these responses with the LPS (lipopolysaccharide)-induced phenotype showed differences in gene expression profiling. PMag positively modulated podosome formation in murine macrophages, but hampered gelatin degradation by these cells. In conclusion, PMag induced an M1-like phenotype that was partially dependent on both TLR4 and ROS. These results show the adjuvant potential of PMag and suggest their use in vaccination schedules.

Effects of salinity and nitrogen supply on the quality and health-related compounds of strawberry fruits (Fragaria × ananassa cv. Primoris).

BACKGROUND: Different nitrogen inputs and/or development under adverse water conditions (water stress/low quality and/or high salinity/electrical conductivity), such as those prevailing in Almeria (Mediterranean coast, south-east Spain), may affect overall fruit and vegetable quality. This study evaluated the influence of salinity and nitrogen reduction in hydroponic nutrient solution on strawberry fruit quality and nutritional compounds (Fragaria × ananassa Duch., cv. Primoris). RESULTS: Strawberries obtained under salinity treatments recorded the highest values for soluble solids content (SSC; all samplings); fruit taste was thus enhanced. Additionally, salinity improved fruit nutritional value, with higher contents of antioxidants compounds (first sampling). During first and second samplings, strawberries grown under N reduction and non-saline conditions showed higher values for firmness compared to fruits developed under other treatments. Regarding health-related compounds, few differences were found except for total polyphenols concentration and antioxidant activity for the first sampling, where strawberries grown under saline treatments obtained the highest values for both parameters. CONCLUSION: The use of low-quality waters, such as those found in Almeria (salinity, N9S and N5S) and low nitrogen inputs (N5, avoid environmental impact) for strawberry cultivation does not exert a negative impact on overall quality. Positive differences could be found in SSC, firmness and health-related compounds when compared against the control treatment (N9).

Shoc2/Sur8 protein regulates neurite outgrowth.

The Shoc2 protein has been implicated in the positive regulation of the Ras-ERK pathway by increasing the functional binding interaction between Ras and Raf, leading to increased ERK activity. Here we found that Shoc2 overexpression induced sustained ERK phosphorylation, notably in the case of EGF stimulation, and Shoc2 knockdown inhibited ERK activation. We demonstrate that ectopic overexpression of human Shoc2 in PC12 cells significantly promotes neurite extension in the presence of EGF, a stimulus that induces proliferation rather than differentiation in these cells. Finally, Shoc2 depletion reduces both NGF-induced neurite outgrowth and ERK activation in PC12 cells. Our data indicate that Shoc2 is essential to modulate the Ras-ERK signaling outcome in cell differentiation processes involved in neurite outgrowth.

Vaccination with recombinant adenoviruses expressing the peste des petits ruminants virus F or H proteins overcomes viral immunosuppression and induces protective immunity against PPRV challenge in sheep.

Peste des petits ruminants (PPR) is a highly contagious disease of small ruminants caused by the Morbillivirus peste des petits ruminants virus (PPRV). Two recombinant replication-defective human adenoviruses serotype 5 (Ad5) expressing either the highly immunogenic fusion protein (F) or hemagglutinin protein (H) from PPRV were used to vaccinate sheep by intramuscular inoculation. Both recombinant adenovirus vaccines elicited PPRV-specific B- and T-cell responses. Thus, neutralizing antibodies were detected in sera from immunized sheep. In addition, we detected a significant antigen specific T-cell response in vaccinated sheep against two different PPRV strains, indicating that the vaccine induced heterologous T cell responses. Importantly, no clinical signs and undetectable virus shedding were observed after virulent PPRV challenge in vaccinated sheep. These vaccines also overcame the T cell immunosuppression induced by PPRV in control animals. The results indicate that these adenovirus constructs could be a promising alternative to current vaccine strategies for the development of PPRV DIVA vaccines.

Inhibition of PI3Kδ reduces kidney infiltration by macrophages and ameliorates systemic lupus in the mouse.

Systemic lupus erythematosus (SLE) is a human chronic inflammatory disease generated and maintained throughout life by autoreactive T and B cells. Class I phosphoinositide 3-kinases (PI3K) are heterodimers composed of a regulatory and a catalytic subunit that catalyze phosphoinositide-3,4,5-P3 formation and regulate cell survival, migration, and division. Activity of the PI3Kδ isoform is enhanced in human SLE patient PBLs. In this study, we analyzed the effect of inhibiting PI3Kδ in MRL/lpr mice, a model of human SLE. We found that PI3Kδ inhibition ameliorated lupus progression. Treatment of these mice with a PI3Kδ inhibitor reduced the excessive numbers of CD4(+) effector/memory cells and B cells. In addition, this treatment reduced serum TNF-α levels and the number of macrophages infiltrating the kidney. Expression of inactive PI3Kδ, but not deletion of the other hematopoietic isoform PI3Kγ, reduced the ability of macrophages to cross the basement membrane, a process required to infiltrate the kidney, explaining MRL/lpr mice improvement by pharmacologic inhibition of PI3Kδ. The observations that p110δ inhibitor prolonged mouse life span, reduced disease symptoms, and showed no obvious secondary effects indicates that PI3Kδ is a promising target for SLE.

Two replication-defective adenoviral vaccine vectors for the induction of immune responses to PPRV.

Peste des petits ruminants is a highly contagious disease of small ruminants caused by a Morbillivirus, peste des petits ruminants virus (PPRV). Two recombinant replication-defective human adenovirus serotype 5 (Ad5) containing the highly immunogenic fusion protein (F) and hemaglutinine protein (H) genes from PPRV were constructed. HEK293A cells infected with either virus (Ad5-PPRV-F or -H) express F and H proteins respectively. These viruses were used to vaccinate mice by intramuscular inoculation. Both viruses elicited PPRV-specific B- and T-cell responses. Thus, after two immunizations, sera from immunized mice elicited neutralizing antibody response, indicating that this approach has the potential to confer protective immunity. In addition, we detected a significant antigen specific CD4(+) and CD8(+) T-cell response in mice vaccinated with either virus. These results indicate that these adenovirus constructs offer a promising alternative to current vaccine strategies for the development of PPRV DIVA vaccines.

Intersectin 1 enhances Cbl ubiquitylation of epidermal growth factor receptor through regulation of Sprouty2-Cbl interaction.

Ubiquitylation of receptor tyrosine kinases plays a critical role in regulating the trafficking and lysosomal degradation of these important signaling molecules. We identified the multidomain scaffolding protein intersectin 1 (ITSN1) as an important regulator of this process (N. P. Martin et al., Mol. Pharmacol. 70:1463-1653, 2006) ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity was unclear. In this study, we found that ITSN1 enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (Spry2) inhibitory protein. We demonstrate that ITSN1 binds Pro-rich regions in both Cbl and Spry2 and that interaction of ITSN1 with Spry2 disrupts Spry2-Cbl interaction, resulting in enhanced ubiquitylation of the EGFR. Disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 results in enhanced Cbl-Spry2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN1 enhances Cbl activity by modulating the interaction of Cbl with Spry2. In addition, our results reveal a new level of complexity in the regulation of Cbl through the interaction with ITSN1 and Spry2.

Mammalian son of sevenless Guanine nucleotide exchange factors: old concepts and new perspectives.

The Son of Sevenless (Sos) factors were originally discovered 2 decades ago as specialized Ras activators in signaling pathways controlling the process of R7 cell development in the eye of Drosophila melanogaster. The 2 known members of the mammalian Sos family (Sos1 and Sos2) code for ubiquitously expressed, highly homologous (69% overall) proteins involved in coupling signals originated by cell surface receptor tyrosine kinases (RTKs) to downstream, Ras-dependent mitogenic signaling pathways. Mechanistically, the Sos proteins function as enzymatic factors interacting with Ras proteins in response to upstream stimuli to promote guanine nucleotide exchange (GDP/GTP) and subsequent formation of the active Ras-GTP complex. In this review, we summarize current knowledge on structural, regulatory, and functional aspects of the Sos family, focusing on specific aspects of Sos biology such as structure-function relationship, crosstalk with different signaling pathways, and in vivo functional significance as deduced from phenotypic characterization of Sos knockout mice and human genetic syndromes caused by germline hSos1 mutations.

Sprouty2 and Spred1-2 proteins inhibit the activation of the ERK pathway elicited by cyclopentenone prostanoids.

Sprouty and Spred proteins have been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. In considering the functional role of these proteins, we explored their effects on ERK activation induced by cyclopentenone prostanoids, which bind to and activate Ras proteins. We therefore found that ectopic overexpression in HeLa cells of human Sprouty2, or human Spred1 or 2, inhibits ERK1/2 and Elk-1 activation triggered by the cyclopentenone prostanoids PGA(1) and 15d-PGJ(2). Furthermore, we found that in HT cells that do not express Sprouty2 due to hypermethylation of its gene-promoter, PGA(1)-provoked ERK activation was more intense and sustained compared to other hematopoietic cell lines with unaltered Sprouty2 expression. Cyclopentenone prostanoids did not induce Sprouty2 tyrosine phosphorylation, in agreement with its incapability to activate tyrosine-kinase receptors. However, Sprouty2 Y55F, which acts as a defective mutant upon tyrosine-kinase receptor stimulation, did not inhibit cyclopentenone prostanoids-elicited ERK pathway activation. In addition, Sprouty2 did not affect the Ras-GTP levels promoted by cyclopentenone prostanoids. These results unveil both common and differential features in the activation of Ras-dependent pathways by cyclopentenone prostanoids and growth factors. Moreover, they provide the first evidence that Sprouty and Spred proteins are negative regulators of the ERK/Elk-1 pathway activation induced not only by growth-factors, but also by reactive lipidic mediators.