Evidence That Ca2+ within the Microdomain of the L-Type Voltage Gated Ca2+ Channel Activates ERK in MIN6 Cells in Response to Glucagon-Like Peptide-1

Glucagon like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient ingestion and acts upon pancreatic β-cells potentiating glucose-stimulated insulin secretion and stimulating β-cell proliferation, differentiation, survival and gene transcription. These effects are mediated through the activation of multiple signal transduction pathways including the extracellular regulated kinase (ERK) pathway. We have previously reported that GLP-1 activates ERK through a mechanism dependent upon the influx of extracellular Ca2+ through L-type voltage gated Ca2+ channels (VGCC). However, the mechanism by which L-type VGCCs couple to the ERK signalling pathway in pancreatic β-cells is poorly understood. In this report, we characterise the relationship between L-type VGCC mediated changes in intracellular Ca2+ concentration ([Ca2+]i) and the activation of ERK, and demonstrate that the sustained activation of ERK (up to 30 min) in response to GLP-1 requires the continual activation of the L-type VGCC yet does not require a sustained increase in global [Ca2+]i or Ca2+ efflux from the endoplasmic reticulum. Moreover, sustained elevation of [Ca2+]i induced by ionomycin is insufficient to stimulate the prolonged activation of ERK. Using the cell permeant Ca2+ chelators, EGTA-AM and BAPTA-AM, to determine the spatial dynamics of L-type VGCC-dependent Ca2+ signalling to ERK, we provide evidence that a sustained increase in Ca2+ within the microdomain of the L-type VGCC is sufficient for signalling to ERK and that this plays an important role in GLP-1- stimulated ERK activation

Effect of in vitro gastrointestinal digestion on the total phenolic contents and antioxidant activity of wild Mediterranean edible plant extracts

The recent interest in wild edible plants is associated with their health benefits, which are mainly due to their richness in antioxidant compounds, particularly phenolics. Nevertheless, some of these compounds are metabolized after ingestion, being transformed into metabolites frequently with lower antioxidant activity. The aim of the present study was to evaluate the influence of the digestive process on the total phenolic contents and antioxidant activity of extracts from four wild edible plants used in the Mediterranean diet (Beta maritima L., Plantago major L., Oxalis pes-caprae L. and Scolymus hispanicus L.). HPLC-DAD analysis revealed that S. hispanicus is characterized by the presence of caffeoylquinic acids, dicaffeoylquinic acids and flavonol derivatives, P. major by high amounts of verbascoside, B. maritima possesses 2,4-dihydroxybenzoic acid, 5-O-caffeoylquinic acid, quercetin derivatives and kaempferol-3-O-rutinoside, and O. pes-caprae extract contains hydroxycinnamic acids and flavone derivatives. Total phenolic contents were determined by Folin-Ciocalteu assay, and antioxidant activity by the ABTS, DPPH, ORAC and FRAP assays. Phenolic contents of P. major and S. hispanicus extracts were not affected by digestion, but they significantly decreased in B. maritima after both phases of digestion process and in O. pes-caprae after the gastric phase. The antioxidant activity results varied with the extract and the method used to evaluate the activity. Results showed that P. major extract has the highest total phenolic contents and antioxidant activity, with considerable values even after digestion, reinforcing the health benefits of this species.European Union (FEDER funds through COMPETE)European Union (EU)European Union (FEDER)European Union (EU)Programa de Cooperacion Interreg V-A Espana - Portugal (POCTEP) 2014-2020 [0377_IBERPHENOL_6_E]project INTERREG - MD. Net: When Brand Meets PeopleFCT Portuguese Foundation for Science and Technolog

Withanolides and related steroids

Since the isolation of the first withanolides in the mid-1960s, over 600 new members of this group of compounds have been described, with most from genera of the plant family Solanaceae. The basic structure of withaferin A, a C28 ergostane with a modified side chain forming a δ-lactone between carbons 22 and 26, was considered for many years the basic template for the withanolides. Nowadays, a considerable number of related structures are also considered part of the withanolide class; among them are those containing γ-lactones in the side chain that have come to be at least as common as the δ-lactones. The reduced versions (γ and δ-lactols) are also known. Further structural variations include modified skeletons (including C27 compounds), aromatic rings and additional rings, which may coexist in a single plant species. Seasonal and geographical variations have also been described in the concentration levels and types of withanolides that may occur, especially in the Jaborosa and Salpichroa genera, and biogenetic relationships among those withanolides may be inferred from the structural variations detected. Withania is the parent genus of the withanolides and a special section is devoted to the new structures isolated from species in this genus. Following this, all other new structures are grouped by structural types. Many withanolides have shown a variety of interesting biological activities ranging from antitumor, cytotoxic and potential cancer chemopreventive effects, to feeding deterrence for several insects as well as selective phytotoxicity towards monocotyledoneous and dicotyledoneous species. Trypanocidal, leishmanicidal, antibacterial, and antifungal activities have also been reported. A comprehensive description of the different activities and their significance has been included in this chapter. The final section is devoted to chemotaxonomic implications of withanolide distribution within the Solanaceae. Overall, this chapter covers the advances in the chemistry and biology of withanolides over the last 16 years.Fil: Misico, Rosana Isabel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos Aplicados a la Química Orgánica (i); ArgentinaFil: Nicotra, V.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinario de Biología Vegetal (p); Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Orgánica; ArgentinaFil: Oberti, Juan Carlos María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinario de Biología Vegetal (p); Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Orgánica; ArgentinaFil: Barboza, Gloria Estela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinario de Biología Vegetal (p); Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; ArgentinaFil: Gil, Roberto Ricardo. University Of Carnegie Mellon; Estados UnidosFil: Burton, Gerardo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos Aplicados a la Química Orgánica (i); Argentin

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

AMD-Associated HTRA1 Variants Do Not Influence TGF-beta Signaling in Microglia

Genetic variants of high-temperature requirement A serine peptidase 1 (HTRA1) and age-related maculopathy susceptibility 2 (ARMS2) are associated with age-related macular degeneration (AMD). One HTRA1 single nucleotide polymorphism (SNP) is situated in the promotor region (rs11200638) resulting in increased expression, while two synonymous SNPs are located in exon 1 (rs1049331:C > T, rs2293870:G > T). HtrA1 is known to inhibit transforming growth factor-beta (TGF-beta) signaling, a pathway regulating quiescence of microglia, the resident immune cells of the brain and retina. Microglia-mediated immune responses contribute to AMD pathogenesis. It is currently unclear whether AMD-associated HTRA1 variants influence TGF-beta signaling and microglia phenotypes. Here, we show that an HtrA1 isoform carrying AMD-associated SNPs in exon 1 exhibits increased proteolytic activity. However, when incubating TGF-beta-treated reactive microglia with HtrA1 protein variants, neither the wildtype nor the SNP-associated isoforms changed microglia activation in vitro