P2Y2 receptor promotes intestinal microtubule stabilization and mucosal re‐epithelization in experimental colitis

P2Y₂ receptor expression is increased in intestinal epithelial cells (IECs) during inflammatory bowel diseases (IBDs). In this context, P2Y₂ stimulates PGE₂ release by IECs, suggesting a role in wound healing. For this study, we have used the non‐cancerous IEC‐6 cell line. IEC‐6 cell migration was determined using Boyden chambers and the single‐edged razor blade model of wounding. The receptor was activated using ATP, UTP, or 2‐thioUTP. Pharmacological inhibitors, a blocking peptide, a neutralizing antibody and interfering RNAs were used to characterize the signaling events. Focal adhesions and microtubule (MT) dynamics were determined by immunofluorescence using anti‐vinculin and anti‐acetylated‐α‐tubulin antibodies, respectively. In vivo, the dextran sodium sulfate mouse model of colitis was used to characterize the effects of P2Y₂ agonist 2‐thioUTP on remission. We showed that P2Y₂ increased cell migration and wound closure by recruiting Go protein with the cooperation of integrin α_v. Following P2Y₂ activation, we demonstrated that GSK3β activity was inhibited in response to Akt activation. This leads to MT stabilization and increased number of focal adhesions. In vivo, P2Y₂ activation stimulates remission, as illustrated by a reduction in the disease activity index values and histological scores as compared to control mice. These findings highlight a novel function for this receptor in IECs. They also illustrate that P2Y receptors could be targeted for the development of innovative therapies for the treatment of IBDs. J. Cell. Physiol. 228: 99–109, 2013. © 2012 Wiley Periodicals, Inc.     

Glucose transporter 2 expression is down regulated following P2X7 activation in enterocytes

With the diabetes epidemic affecting the world population, there is an increasing demand for means to regulate glycemia. Dietary glucose is first absorbed by the intestine before entering the blood stream. Thus, the regulation of glucose absorption by intestinal epithelial cells (IECs) could represent a way to regulate glycemia. Among the molecules involved in glycemia homeostasis, extracellular ATP, a paracrine signaling molecule, was reported to induce insulin secretion from pancreatic β cells by activating P2Y and P2X receptors. In rat's jejunum, P2X7 expression was previously immunolocalized to the apex of villi, where it has been suspected to play a role in apoptosis. However, using an antibody recognizing the receptor extracellular domain and thus most of the P2X7 isoforms, we showed that expression of this receptor is apparent in the top two‐thirds of villi. These data suggest a different role for this receptor in IECs. Using the non‐cancerous IEC‐6 cells and differentiated Caco‐2 cells, glucose transport was reduced by more than 30% following P2X7 stimulation. This effect on glucose transport was not due to P2X7‐induced cell apoptosis, but rather was the consequence of glucose transporter 2 (Glut2)'s internalization. The signaling pathway leading to P2X7‐dependent Glut2 internalization involved the calcium‐independent activation of phospholipase Cγ1 (PLCγ1), PKCδ, and PKD1. Although the complete mechanism regulating Glut2 internalization following P2X7 activation is not fully understood, modulation of P2X7 receptor activation could represent an interesting approach to regulate intestinal glucose absorption. J. Cell. Physiol. 228: 120–129, 2013. © 2012 Wiley Periodicals, Inc.     

Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans), an increased tolerance to Cry4Aa (3.5-fold) and Cry11Aa toxins (8-fold) was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.     

Induction of the Cpx Envelope Stress Pathway Contributes to Escherichia coli Tolerance to Antimicrobial Peptides

Antimicrobial peptides produced by multicellular organisms as part of their innate system of defense against microorganisms are currently considered potential alternatives to conventional antibiotics in case of infection by multiresistant bacteria. However, while the mode of action of antimicrobial peptides is relatively well described, resistance mechanisms potentially induced or selected by these peptides are still poorly understood. In this work, we studied the mechanisms of action and resistance potentially induced by ApoEdpL-W, a new antimicrobial peptide derived from human apolipoprotein E. Investigation of the genetic response of Escherichia coli upon exposure to sublethal concentrations of ApoEdpL-W revealed that this antimicrobial peptide triggers activation of RcsCDB, CpxAR, and σ^E envelope stress pathways. This genetic response is not restricted to ApoEdpL-W, since several other antimicrobial peptides, including polymyxin B, melittin, LL-37, and modified S₄ dermaseptin, also activate several E. coli envelope stress pathways. Finally, we demonstrate that induction of the CpxAR two-component system directly contributes to E. coli tolerance toward ApoEdpL-W, polymyxin B, and melittin. These results therefore show that E. coli senses and responds to different antimicrobial peptides by activation of the CpxAR pathway. While this study further extends the understanding of the array of peptide-induced stress signaling systems, it also provides insight into the contribution of Cpx envelope stress pathway to E. coli tolerance to antimicrobial peptides.     

Nest survival of Woodpigeons (Columba palumbus) in North African forests

Capsule The nesting success of the Woodpigeon Columba palumbus in oak forests in Morocco is affected by human disturbance and nest-site characteristics.

Aims To determine factors affecting the nesting success of Woodpigeons subject to two levels of disturbance in a holm oak Middle Atlas forest.

Methods Woodpigeon nests were monitored in highly disturbed (n?=?30) and less-disturbed sites (n?=?60) over two years (2010–11). Logistic-exposure models were used to estimate daily nest-survival rate and evaluate mechanisms (altitude, temporal factors, nest location, nest-site habitat and degree of human influence) affecting nest survival.

Results Daily nest-survival rate was higher in less-disturbed sites (0.987; 95% CL: 0.980–0.991) than in high-disturbance sites (0.967; 95% CL: 0.949–0.978). Increasing density of trees surrounding nest sites and distance from the nest tree to the closest track increased daily nest-survival rate.

Conclusions Less-disturbed sites confer better nesting conditions than highly disturbed ones. Management measures should control the access of vehicles, especially trucks, in the forest of the Middle Atlas and introduce a grazing regime that will allow the forest regeneration, which would improve Woodpigeon nesting success by increasing the density of holm oak trees.     

Factors affecting reproduction of Woodpigeons Columba palumbus in North African forests: 1. Nest habitat selection

Capsule Woodpigeon Columba palumbus nest-tree selection is affected by human disturbance, grazing and tree characteristics.

Aims To determine the characteristics of trees used for nesting by Woodpigeons under two levels of grazing and disturbance in a Holm oak Middle Atlas forest, Morocco.

Methods Woodpigeon nest-tree selection was investigated in highly disturbed and less-disturbed sites over two years (2010 and 2011). The features of nest-tree characteristics were determined by comparing the characteristics of trees used for nesting to those of randomly selected trees.

Results Tree physical characteristics, grazing intensity and level of human disturbance were the main factors determining selection for nest-trees. Woodpigeons chose nesting trees that were greater in height, larger in DBH, lower in base of the live crown, in denser stands and at greater distance from tracks.

Conclusions Forest management for Woodpigeon nest habitat selection should focus on controlling the access of vehicles especially trucks, introducing a rotating system of forest utilization for grazing and maintaining (without extending) the cereal crops. These recommendations may assist land managers to plan for continued persistence of this species in Middle Atlas forests.     

Functional conservation between mammalian MGRN1 and plant LOG2 ubiquitin ligases

Plant LOSS OF GDU 2 (LOG2) and Mammalian Mahogunin Ring Finger 1 (MGRN1) proteins are RING-type E3 ligases sharing similarity N-terminal to the RING domain. Deletion of this region disrupts the interaction of LOG2 with the plant membrane protein GLUTAMINE DUMPER1 (GDU1). Phylogenetic analysis identified two clades of LOG2/MGRN1-like proteins in vertebrates and plants. The ability of MGRN1 to functionally replace LOG2 was tested. MGRN1 ubiquitylates GDU1 in vitro and can partially substitute for LOG2 in the plant, partially restoring amino acid resistance to a GDU1-myc over-expression, log2-2 background. Altogether, these results suggest a conserved function for the N-terminal domain in evolution.     

A root chicory MADS box sequence and the Arabidopsis flowering repressor FLC share common features that suggest conserved function in vernalization and de‐vernalization responses

Root chicory (Cichorium intybus var. sativum) is a biennial crop, but is harvested to obtain root inulin at the end of the first growing season before flowering. However, cold temperatures may vernalize seeds or plantlets, leading to incidental early flowering, and hence understanding the molecular basis of vernalization is important. A MADS box sequence was isolated by RT‐PCR and named FLC‐LIKE1 (CiFL1) because of its phylogenetic positioning within the same clade as the floral repressor Arabidopsis FLOWERING LOCUS C (AtFLC). Moreover, over‐expression of CiFL1 in Arabidopsis caused late flowering and prevented up‐regulation of the AtFLC target FLOWERING LOCUS T by photoperiod, suggesting functional conservation between root chicory and Arabidopsis. Like AtFLC in Arabidopsis, CiFL1 was repressed during vernalization of seeds or plantlets of chicory, but repression of CiFL1 was unstable when the post‐vernalization temperature was favorable to flowering and when it de‐vernalized the plants. This instability of CiFL1 repression may be linked to the bienniality of root chicory compared with the annual lifecycle of Arabidopsis. However, re‐activation of AtFLC was also observed in Arabidopsis when a high temperature treatment was used straight after seed vernalization, eliminating the promotive effect of cold on flowering. Cold‐induced down‐regulation of a MADS box floral repressor and its re‐activation by high temperature thus appear to be conserved features of the vernalization and de‐vernalization responses in distant species.     

Pleiotropy in the melanocortin system: expression levels of this system are associated with melanogenesis and pigmentation in the tawny owl (Strix aluco)

The adaptive function of melanin‐based coloration is a long‐standing debate. A recent genetic model suggested that pleiotropy could account for covariations between pigmentation, behaviour, morphology, physiology and life history traits. We explored whether the expression levels of genes belonging to the melanocortin system (MC1R, POMC, PC1/3, PC2 and the antagonist ASIP), which have many pleiotropic effects, are associated with melanogenesis (through variation in the expression of the genes MITF, SLC7A11, TYR, TYRP1) and in turn melanin‐based coloration. We considered the tawny owl (Strix aluco) because individuals vary continuously from light to dark reddish, and thus, colour variation is likely to stem from differences in the levels of gene expression. We measured gene expression in feather bases collected in nestlings at the time of melanin production. As expected, the melanocortin system was associated with the expression of melanogenic genes and pigmentation. Offspring of darker reddish fathers expressed PC1/3 to lower levels but tended to express PC2 to higher levels. The convertase enzyme PC1/3 cleaves the POMC prohormone to obtain ACTH, while the convertase enzyme PC2 cleaves ACTH to produce α‐melanin‐stimulating hormone (α‐MSH). ACTH regulates glucocorticoids, hormones that modulate stress responses, while α‐MSH induces eumelanogenesis. We therefore conclude that the melanocortin system, through the convertase enzymes PC1/3 and PC2, may account for part of the interindividual variation in melanin‐based coloration in nestling tawny owls. Pleiotropy may thus account for the covariation between phenotypic traits involved in social interactions (here pigmentation) and life history, morphology, behaviour and physiology.