Bicyclic Peptide Based Lectinomimic

Peptide based lectin mimetics represent an attractive approach for the development of artificial carbohydrate receptors that might find application in bio-analytical and medicinal fields. Taking into consideration the structure of typical lectin binding site, we have designed a novel artificial receptor molecule possessing a rigid three-dimensional structure, hydrogen-bonding site and lipophilic binding pocket to promote hydrophobic interaction and hydrogen-bonding. A new solid- phase synthetic approach that allows complete synthesis of desired bicyclic peptide 1 on the solid support was developed. CD spectra of peptides 1 and 2 indicate that the structure of 1 is rather rigid and preorganised for the three-dimensional monosaccharide substrates binding. The binding affinities of bicyclic peptide receptor 1 toward various carbohydrate substrates at physiologically relevant conditions were estimated by UV/vis and fluorimetric titration experiments, and the observed values are in the millimolar range. With these results we have demonstrated that the bicyclic peptide 1 represent a promising basis for the design of new and more efficient carbohydrate receptors that may have broader application in bio-analytical or medicinal field

Butterfly distribution along altitudinal gradients: temporal changes over a short time period

Mountain ecosystems are particularly sensitive to changes in climate and land cover, but at the same time, they can offer important refuges for species on the opposite of the more altered lowlands. To explore the potential role of mountain ecosystems in butterfly conservation and to assess the vulnerability of the alpine species, we analyzed the short-term changes (2006-2008 vs. 2012-2013) of butterflies\u27 distribution along altitudinal gradients in the NW Italian Alps. We sampled butterfly communities once a month (62 sampling stations, 3 seasonal replicates per year, from June to August) by semi-quantitative sampling techniques. The monitored gradient ranges from the montane to the alpine belt (600-2700 m a.s.l.) within three protected areas: Gran Paradiso National Park (LTER, Sitecode: LTER_EU_IT_109), Orsiera Rocciavr? Natural Park and Veglia Devero Natural Park. We investigated butterflies\u27 temporal changes in accordance with a hierarchical approach to assess potential relationships between species and community level. As a first step, we characterized each species in terms of habitat requirements, elevational range and temperature preferences and we compared plot occupancy and altitudinal range changes between time periods (2006-2008 vs. 2012-2013). Secondly, we focused on community level, analyzing species richness and community composition temporal changes. The species level analysis highlighted a general increase in mean occupancy level and significant changes at both altitudinal boundaries. Looking at the ecological groups, we observed an increase of generalist and highly mobile species at the expense of the specialist and less mobile ones. For the community level, we noticed a significant increase in species richness, in the community temperature index and a tendency towards homogenization within communities. Besides the short time period considered, butterflies species distribution and communities changed considerably. In light of these results, it is fundamental to continue monitoring activities to understand if we are facing transient changes or first signals of an imminent trend

Functional analysis of two-amino acid substitutions in gp91 phox in a patient with X-linked flavocytochrome b558-positive chronic granulomatous disease by means of transgenic PLB-985 cells

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. The most common form is caused by mutations in the CYBB gene encoding gp91phox protein, the heavy chain of cytochrome b558, which is the redox element of NADPH oxidase. In some rare cases, the mutated gp91phox is normally expressed but no NADPH oxidase can be detected. This type of CGD is called X91+ CGD. We have previously reported an X+ CGD case with a double-missense mutation in gp91phox. Transgenic PLB-985 cells have now been made to study the impact of each single mutation on oxidase activity and assembly to rule out a possible new polymorphism in the CYBB gene. The His303Asn/Pro304Arg gp91phox transgenic PLB-985 cells exactly mimic the phenotype of the neutrophils of the X+ CGD patient. The His303Asn mutation is sufficient to inhibit oxidase activity in intact cells and in a broken cell system, whereas in the Pro304Arg mutant, residual activity suggests that the Pro304Arg substitution is less devastating to oxidase activity than the His303Asn mutation. The study of NADPH oxidase assembly following the in vitro and in vivo translocation of cytosolic factors p47phox and p67phox has demonstrated that, in the double mutant and in the His303Asn mutant, NADPH oxidase assembly is abolished, although the translocation is only attenuated in Pro304Arg mutant cells. Thus, even though the His303Asn mutation has a more severe inhibitory effect on NADPH oxidase activity and assembly than the Pro304Arg mutation, neither mutation can be considered as a polymorphism

Diet of tadpoles of Physalaemus biligonigerus (Leiuperidae) from agricultural ponds in the central region of Argentina

The intensification of agriculture has led an important loss of natural habitats, with significant consequences for biodiversity. In this sense, the studies on anuran amphibian tadpoles inhabiting these environments are relevant, because the larval stage is a phase of population regulation. The aim of this study was to analyze the diet in Physalaemus biligonigerus tadpoles, an anuran species widely distributed in South America and that inhabit agroecosystems. Three sites were sampled; two agroecosystems with different alteration degrees (AG1 and AG2) and an uncultured (SM) third place. The captured tadpoles were anesthetized, fixed and preserved in formaldehyde (10%). Subsequently, the complete intestine was removed and analyzed for food items under a binocular microscope. The diet in P. biligonigerus tadpoles has a dominance of algae Bacillariophyceae, mainly in agroecosystems, due to the presence of the genera Navicula, Nitzschia and Gomphonema. There was a considerable abundance of the Gomphonema genus in the AG2 site. In addition, in the AG1 site several non-diatom algae were particularly abundant in the diet, such as the genera Euglena, Oedogonium and Chaetophora. In the SM site, the non-diatom genus Oscillatoria was well represented in the diet. Tadpoles inhabiting the site with abundant crop and livestock (AG1) ingested a significantly smaller amount of food. The presence of certain algae associated with eutrophic environments could indicate some pollution in agroecosystems (AG1 and AG2). Larval diet is suggested as a potential bioindicator of environmental health for these areas

Transient Alteration of Cellular Redox Buffering before Irradiation Triggers Apoptosis in Head and Neck Carcinoma Stem and Non-Stem Cells

Background: Head and neck squamous cell carcinoma (HNSCC) is an aggressive and recurrent malignancy owing to intrinsic radioresistance and lack of induction of apoptosis. The major focus of this work was to design a transient glutathione depleting strategy during the course of irradiation of HNSCC in order to overcome their radioresistance associated with redox adaptation. Methodology/Principal Findings: Treatment of SQ20B cells with dimethylfumarate (DMF), a GSH-depleting agent, and L-Buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis 4 h before a 10 Gy irradiation led to the lowering of the endogenous GSH content to less than 10 % of that in control cells and to the triggering of radiation-induced apoptotic cell death. The sequence of biochemical events after GSH depletion and irradiation included ASK-1 followed by JNK activation which resulted in the triggering of the intrinsic apoptotic pathway through Bax translocation to mitochondria. Conclusions: This transient GSH depletion also triggered radiation-induced cell death in SQ20B stem cells, a key event to overcome locoregional recurrence of HNSCC. Finally, our in vivo data highlight the relevance for further clinical trials o

The Targeting of Plasmalemmal Ceramide to Mitochondria during Apoptosis

Ceramide is a key lipid mediator of cellular processes such as differentiation, proliferation, growth arrest and apoptosis. During apoptosis, ceramide is produced within the plasma membrane. Although recent data suggest that the generation of intracellular ceramide increases mitochondrial permeability, the source of mitochondrial ceramide remains unknown. Here, we determine whether a stress-mediated plasmalemmal pool of ceramide might become available to the mitochondria of apoptotic cells. We have previously established annexin A1—a member of a family of Ca2+ and membrane-binding proteins—to be a marker of ceramide platforms. Using fluorescently tagged annexin A1, we show that, upon its generation within the plasma membrane, ceramide self-associates into platforms that subsequently invaginate and fuse with mitochondria. An accumulation of ceramide within the mitochondria of apoptotic cells was also confirmed using a ceramide-specific antibody. Electron microscopic tomography confirmed that upon the formation of ceramide platforms, the invaginated regions of the plasma membrane extend deep into the cytoplasm forming direct physical contacts with mitochondrial outer membranes. Ceramide might thus be directly transferred from the plasma membrane to the mitochondrial outer membrane. It is conceivable that this “kiss-of-death” increases the permeability of the mitochondrial outer membrane thereby triggering apoptosis

The role of the mitochondria and the endoplasmic reticulum contact sites in the development of the immune responses

Abstract Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are dynamic modules enriched in subset of lipids and specialized proteins that determine their structure and functions. The MERCs regulate lipid transfer, autophagosome formation, mitochondrial fission, Ca2+ homeostasis and apoptosis. Since these functions are essential for cell biology, it is therefore not surprising that MERCs also play a critical role in organ physiology among which the immune system stands by its critical host defense function. This defense system must discriminate and tolerate host cells and beneficial commensal microorganisms while eliminating pathogenic ones in order to preserve normal homeostasis. To meet this goal, the immune system has two lines of defense. First, the fast acting but unspecific innate immune system relies on anatomical physical barriers and subsets of hematopoietically derived cells expressing germline-encoded receptors called pattern recognition receptors (PRR) recognizing conserved motifs on the pathogens. Second, the slower but very specific adaptive immune response is added to complement innate immunity. Adaptive immunity relies on another set of specialized cells, the lymphocytes, harboring receptors requiring somatic recombination to be expressed. Both innate and adaptive immune cells must be activated to phagocytose and process pathogens, migrate, proliferate, release soluble factors and destroy infected cells. Some of these functions are strongly dependent on lipid transfer, autophagosome formation, mitochondrial fission, and Ca2+ flux; this indicates that MERCs could regulate immunity