Effects of dietary omega-6 and omega-3 fatty acids on levels of long chain polyunsaturated fatty acids in erythrocytes]

C18:2 omega 6/C18:3 omega 3 ratio was lowered in the diet of Elderly subjects. This was done by the replacement of usual sunflower oil by rapeseed oil or by supplementing soybean oil. This diet modification induced an increase of EPA (C20:5 omega 3) and DHA (C22:6 omega 3) in red cell phospholipids. The omega 6 fatty acids (C18:2 and C20:4) were slightly modified. Therefore, dietary C18:2 omega 6/C18:3 omega 3 ratio, seems to play an important role in the determination of membrane highly unsaturated fatty acid levels.     

Square‐Centimeter‐Sized High‐Efficiency Polymer Solar Cells: How the Processing Atmosphere and Film Quality Influence Performance at Large Scale

Organic solar cells based on two benzodithiophene‐based polymers (PTB7 and PTB7‐Th) processed at square centimeter‐size under inert atmosphere and ambient air, respectively, are investigated. It is demonstrated that the performance of solar cells processed under inert atmosphere is not limited by the upscaling of photoactive layer and the interfacial layers. Thorough morphological and electrical characterizations of optimized layers and corresponding devices reveal that performance losses due to area enlargement are only caused by the sheet resistance of the transparent electrode reducing the efficiency from 9.3% of 7.8% for PTB7‐Th in the condition that both photoactive layer and the interfacial layers are of high layer quality. Air processing of photoactive layer and the interfacial layers into centimeter‐sized solar cells lead to additional, but only slight, losses (<10%) in all photovoltaic parameters, which can be addressed to changes in the electronic properties of both active layer and ZnO layers rather than changes in layer morphology. The demonstrated compatibility of polymer solar cells using solution‐processed photoactive layer and interfacial layers with large area indicates that the introduction of a standard active area of 1 cm² for measuring efficiency of organic record solar cells is feasible. However electric standards for indium tin oxides (ITO) or alternative transparent electrodes need to be developed so that performance of new photovoltaic materials can be compared at square centimeter‐size.     

Unraveling G protein-coupled receptor endocytosis pathways using real-time monitoring of agonist-promoted interaction between beta-arrestins and AP-2

The most widely studied pathway underlying agonist-promoted internalization of G protein-coupled receptors (GPCRs) involves beta-arrestin and clathrin-coated pits. However, both beta-arrestin- and clathrin-independent processes have also been reported. Classically, the endocytic routes are characterized using pharmacological inhibitors and various dominant negative mutants, resulting sometimes in conflicting results and interpretational difficulties. Here, taking advantage of the fact that beta-arrestin binding to the beta2 subunit of the clathrin adaptor AP-2 (beta2-adaptin) is needed for the beta-arrestin-mediated targeting of GPCRs to clathrin-coated pits, we developed a bioluminescence resonance energy transfer-based approach directly assessing the molecular steps involved in the endocytosis of GPCRs in living cells. For 10 of the 12 receptors tested, including some that were previously suggested to internalize via clathrin-independent pathways, agonist stimulation promoted beta-arrestin 1 and 2 interaction with beta2-adaptin, indicating a beta-arrestin- and clathrin-dependent endocytic process. Detailed analyses of beta-arrestin interactions with both the receptor and beta2-adaptin also allowed us to demonstrate that recruitment of beta-arrestins to the receptor and the ensuing conformational changes are the leading events preceding AP-2 engagement and subsequent clathrin-mediated endocytosis. Among the receptors tested, only the endothelin A and B receptors failed to promote interaction between beta-arrestins and beta2-adaptin. However, both receptors recruited beta-arrestins upon agonist stimulation, suggesting a beta-arrestin-dependent but clathrin-independent route of internalization for these two receptors. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the bioluminescence resonance energy transfer-based beta-arrestin/beta2-adaptin interaction assay represents a novel biosensor to assess receptor activation.     

Curcumin attenuates proangiogenic and proinflammatory factors in human eutopic endometrial stromal cells through the NF-κB signaling pathway

Endometriosis is a chronic gynecological inflammatory disorder in which immune system dysregulation is thought to play a role in its initiation and progression. Due to altered sex steroid receptor concentrations and other signaling defects, eutopic endometriotic tissues have an attenuated response to progesterone. This progesterone-resistance contributes to lesion survival, proliferation, pain, and infertility. The current agency-approved hormonal therapies, including synthetic progestins, GnRH agonists, and danazol are often of limited efficacy and counterproductive to fertility and cause systemic side effects due to suppression of endogenous steroid hormone levels. In the current study, we examined the effects of curcumin (CUR, diferuloylmethane), which has long been used as an anti-inflammatory folk medicine in Asian countries for this condition. The basal levels of proinflammatory and proangiogenic chemokines and cytokines expression were higher in primary cultures of stromal cells derived from eutopic endometrium of endometriosis (EESC) subjects compared with normal endometrial stromal cells (NESC). The treatment of EESC and NESC with CUR significantly and dose-dependently reduced chemokine and cytokine secretion over the time course. Notably, CUR treatment significantly decreased phosphorylation of the IKKα/β, NF-κB, STAT3, and JNK signaling pathways under these experimental conditions. Taken together, our findings suggest that CUR has therapeutic potential to abrogate aberrant activation of chemokines and cytokines, and IKKα/β, NF-κB, STAT3, and JNK signaling pathways to reduce inflammation associated with endometriosis.     

Genome-scale design of PCR primers and long oligomers for DNA microarrays

During the last years, the demand for custom-made cDNA chips/arrays as well as whole genome chips is increasing rapidly. The efficient selection of gene-specific primers/oligomers is of the utmost importance for the successful production of such chips. We developed GenomePRIDE, a highly flexible and scalable software for designing primers/oligomers for large-scale projects. The program is able to generate either long oligomers (40–70 bases), or PCR primers for the amplification of gene-specific DNA fragments of user-defined length. Additionally, primers can be designed in-frame in order to facilitate large-scale cloning into expression vectors. Furthermore, GenomePRIDE can be adapted to specific applications such as the generation of genomic amplicon arrays or the design of fragments specific for alternative splice isoforms. We tested the performance of GenomePRIDE on the entire genomes of Listeria monocytogenes (1584 gene-specific PCRs, 48 long oligomers) as well as of eukaryotes such as Schizosaccharomyces pombe (5006 gene-specific PCRs), and Drosophila melanogaster (21 306 gene-specific PCRs). With its computing speed of 1000 primer pairs per hour and a PCR amplification success of 99%, GenomePRIDE represents an extremely cost- and time-effective program.     

Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation

Nitric oxide (NO) is synthesized from l-arginine by the Ca(2+)/calmodulin-sensitive endothelial NO synthase (NOS) isoform (eNOS). The present study assesses the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) in endothelium-dependent relaxation and NO synthesis. The effects of three CaMK II inhibitors were investigated in endothelium-intact aortic rings of normotensive rats. NO synthesis was assessed by a NO sensor and chemiluminescence in culture medium of cultured porcine aortic endothelial cells stimulated with the Ca(2+) ionophore A23187 and thapsigargin. Rat aortic endothelial NOS activity was measured by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline. Three CaMK II inhibitors, polypeptide 281-302, KN-93, and lavendustin C, attenuated the endothelium-dependent relaxation of endothelium-intact rat aortic rings in response to acetylcholine, A23187, and thapsigargin. None of the CaMK II inhibitors affected the relaxation induced by NO donors. In a porcine aortic endothelial cell line, KN-93 decreased NO synthesis and caused a rightward shift of the concentration-response curves to A23187 and thapsigargin. In rat aortic endothelial cells, KN-93 significantly decreased bradykinin-induced eNOS activity. These results suggest that CaMK II was involved in NO synthesis as a result of Ca(2+)-dependent activation of eNOS.     

Alpha 1-adrenergic and cholinergic agonists activate MAPK by separate mechanisms to inhibit secretion in lacrimal gland

Thepurpose of this study was to determine the role of p42/p44mitogen-activated protein kinase (MAPK) in₁-adrenergically and cholinergically stimulated proteinsecretion in rat lacrimal gland acinar cells and the pathways used bythese agonists to activate MAPK. Acini were isolated by collagenasedigestion and incubated with the ₁-adrenergic agonistphenylephrine or the cholinergic agonist carbachol, and activation ofMAPK and protein secretion were then measured. Phenylephrine andcarbachol activated MAPK in a time- and concentration-dependent manner.Inhibition of MAPK significantly increased phenylephrine- andcarbachol-induced protein secretion. Inhibition of EGF receptor (EGFR)with AG1478, an inhibitor of the EGFR tyrosine kinase activity,significantly increased phenylephrine- but not carbachol-inducedprotein secretion. Whereas phenylephrine-induced activation of MAPK wascompletely inhibited by AG1478, activation of MAPK by carbachol wasnot. Phenylephrine stimulated tyrosine phosphorylation of the EGFR, whereas carbachol stimulated p60^Src, and possibly Pyk2, toactivate MAPK. We conclude that, in the lacrimal gland, activation ofMAPK plays an inhibitory role in ₁-adrenergically andcholinergically stimulated protein secretion and that these agonistsuse different signaling mechanisms to activate MAPK.

     

Galphaq-coupled receptor internalization specifically induced by Galphaq signaling. Regulation by EBP50

In the present report, we investigated the effect of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) expression on the agonist-induced internalization of the thromboxane A(2) beta receptor (TPbeta receptor). Interestingly, we found that EBP50 almost completely blocked TPbeta receptor internalization, which could not be reversed by overexpression of G protein-coupled receptor (GPCR) kinases and arrestins. Because we recently demonstrated that EBP50 can bind to and inhibit Galpha(q), we next studied whether Galpha(q) signaling could induce TPbeta receptor internalization, addressing the long standing question about the relationship between GPCR signaling and their internalization. Expression of a constitutively active Galpha(q) mutant (Galpha(q)-R183C) resulted in a robust internalization of the TPbeta receptor, which was unaffected by expression of dominant negative mutants of arrestin-2 and -3, but inhibited by expression of EBP50 or dynamin-K44A, a dominant negative mutant of dynamin. Phospholipase Cbeta and protein kinase C did not appear to significantly contribute to internalization of the TPbeta receptor, suggesting that Galpha(q) induces receptor internalization through a phospholipase Cbeta- and protein kinase C-independent pathway. Surprisingly, there appears to be specificity in Galpha protein-mediated GPCR internalization. Galpha(q)-R183C also induced the internalization of CXCR4 (Galpha(q)-coupled), whereas it failed to do so for the beta(2)-adrenergic receptor (Galpha(s)-coupled). Moreover, Galpha(s)-R201C, a constitutively active form of Galpha(s), had no effect on internalization of the TPbeta, CXCR4, and beta(2)-adrenergic receptors. Thus, we showed that Galpha protein signaling can lead to internalization of GPCRs, with specificity in both the Galpha proteins and GPCRs that are involved. Furthermore, a new function has been described for EBP50 in its capacity to inhibit receptor endocytosis.