A comprehensive review of the influence of Epigallocatechin gallate on Sjögren's syndrome associated molecular regulators of exocytosis (Review).

Sjögren's syndrome (SS) is an autoimmune disorder that affects the salivary glands, leading to reduced secretory functions and oral and ocular dryness. The salivary glands are composed of acinar cells that are responsible for the secretion and production of secretory granules, which contain salivary components, such as amylase, mucins and immunoglobulins. This secretion process involves secretory vesicle trafficking, docking, priming and membrane fusion. A failure during any of the steps in exocytosis in the salivary glands results in the altered secretion of saliva. Soluble N-ethylmaleimide-sensitive-factor attachment protein receptors, actin, tight junctions and aquaporin 5 all serve an important role in the trafficking regulation of secretory vesicles in the secretion of saliva via exocytosis. Alterations in the expression and distribution of these selected proteins leads to salivary gland dysfunction, including SS. Several studies have demonstrated that green tea polyphenols, most notably Epigallocatechin gallate (EGCG), possess both anti-inflammatory and anti-apoptotic properties in normal human cells. Molecular, cellular and animal studies have indicated that EGCG can provide protective effects against autoimmune and inflammatory reactions in salivary glands in diseases such as SS. The aim of the present article is to provide a comprehensive and up-to-date review on the possible therapeutic interactions between EGCG and the selected molecular mechanisms associated with SS.

Adipose-PAS interactions in the context of its localised bio-engineering potential (Review).

Adipocytes are a known source of stem cells. They are easy to harvest, and are a suitable candidate for autogenous grafts. Adipose derived stem cells (ADSCs) have multiple target tissues which they can differentiate into, including bone and cartilage. In adipose tissue, ADSCs are able to differentiate, as well as providing energy and a supply of cytokines/hormones to manage the hypoxic and lipid/hormone saturated adipose environment. The plasminogen activation system (PAS) controls the majority of proteolytic activities in both adipose and wound healing environments, allowing for rapid cellular migration and tissue remodelling. While the primary activation pathway for PAS occurs through the urokinase plasminogen activator (uPA), which is highly expressed by endothelial cells, its function is not limited to enabling revascularisation. Proteolytic activity is dependent on protease activation, localisation, recycling mechanisms and substrate availability. uPA and uPA activated plasminogen allows pluripotent cells to arrive to new local environments and fulfil the niche demands. However, overstimulation, the acquisition of a migratory phenotype and constant protein turnover can be unconducive to the formation of structured hard and soft tissues. To maintain a suitable healing pattern, the proteolytic activity stimulated by uPA is modulated by plasminogen activator inhibitor 1. Depending on the physiological settings, different parts of the remodelling mechanism are activated with varying results. Utilising the differences within each microenvironment to recreate a desired niche is a valid therapeutic bio-engineering approach. By controlling the rate of protein turnover combined with a receptive stem cell lineage, such as ADSC, a novel avenue on the therapeutic opportunities may be identified, which can overcome limitations, such as scarcity of stem cells, low angiogenic potential or poor host tissue adaptation.

The Solanum lycopersicum WRKY3 Transcription Factor SlWRKY3 Is Involved in Salt Stress Tolerance in Tomato.

Salinity threatens productivity of economically important crops such as tomato (Solanum lycopersicum L.). WRKY transcription factors appear, from a growing body of knowledge, as important regulators of abiotic stresses tolerance. Tomato SlWRKY3 is a nuclear protein binding to the consensus CGTTGACC/T W box. SlWRKY3 is preferentially expressed in aged organs, and is rapidly induced by NaCl, KCl, and drought. In addition, SlWRKY3 responds to salicylic acid, and 35S::SlWRKY3 tomatoes showed under salt treatment reduced contents of salicylic acid. In tomato, overexpression of SlWRKY3 impacted multiple aspects of salinity tolerance. Indeed, salinized (125 mM NaCl, 20 days) 35S::SlWRKY3 tomato plants displayed reduced oxidative stress and proline contents compared to WT. Physiological parameters related to plant growth (shoot and root biomass) and photosynthesis (stomatal conductance and chlorophyll a content) were retained in transgenic plants, together with lower Na+ contents in leaves, and higher accumulation of K+ and Ca2+. Microarray analysis confirmed that many stress-related genes were already up-regulated in transgenic tomatoes under optimal conditions of growth, including genes coding for antioxidant enzymes, ion and water transporters, or plant defense proteins. Together, these results indicate that SlWRKY3 is an important regulator of salinity tolerance in tomato.

Female bovine blastocysts are more prone to apoptosis than male ones.

Female and male embryos show differences in gene expression and metabolism from the onset of their genome. Those differences are affected by environmental factors. The objective of the study was to compare the apoptotic rates of in vitro-produced female and male bovine blastocysts cultured in different conditions. Day 7 blastocysts obtained after IVF with sex-sorted semen and culture in two synthetic oviductal fluid-based media (containing fetal calf serum [FCS] or BSA, insulin, transferrin, and selenium) were simultaneously evaluated for two markers of apoptosis after 3D reconstruction from confocal images: active caspase 3 by immunofluorescence and DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling. Higher levels of apoptotic cells were observed in female embryos whatever the culture condition but with a more pronounced difference in FCS medium. This result was confirmed using the unsexed semen of two bulls. The sex effect on apoptosis was detected in both the inner cell mass and the trophectoderm but was dependent on the embryonic size. In conclusion, this study reported that female bovine blastocysts are more prone to apoptosis than male ones but that culture in FCS exacerbates the differences in apoptosis between sexes, especially in small blastocysts.

Infection dynamic of symbiotic bacteria in the pea aphid Acyrthosiphon pisum gut and host immune response at the early steps in the infection process.

In addition to its obligatory symbiont Buchnera aphidicola, the pea aphid Acyrthosiphon pisum can harbor several facultative bacterial symbionts which can be mutualistic in the context of various ecological interactions. Belonging to a genus where many members have been described as pathogen in invertebrates, Serratia symbiotica is one of the most common facultative partners found in aphids. The recent discovery of strains able to grow outside their host allowed us to simulate environmental acquisition of symbiotic bacteria by aphids. Here, we performed an experiment to characterize the A. pisum response to the ingestion of the free-living S. symbiotica CWBI-2.3T in comparison to the ingestion of the pathogenic Serratia marcescens Db11 at the early steps in the infection process. We found that, while S. marcescens Db11 killed the aphids within a few days, S. symbiotica CWBI-2.3T did not affect host survival and colonized the whole digestive tract within a few days. Gene expression analysis of immune genes suggests that S. symbiotica CWBI-2.3T did not trigger an immune reaction, while S. marcescens Db11 did, and supports the hypothesis of a fine-tuning of the host immune response set-up for fighting pathogens while maintaining mutualistic partners. Our results also suggest that the lysosomal system and the JNK pathway are possibly involved in the regulation of invasive bacteria in aphids and that the activation of the JNK pathway is IMD-independent in the pea aphid.

Effect of polyunsaturated fatty acids on tight junctions in a model of the human intestinal epithelium under normal and inflammatory conditions.

Owing to their immune-modulatory action on the intestinal mucosa immune cells, the n-3 and n-6 polyunsaturated fatty acids (PUFA) have been suggested to modulate the risk and development of inflammatory bowel diseases. Failure in the intestinal barrier is an important hallmark of inflammatory bowel diseases. This study aimed at evaluating the impact of dietary PUFA on tight junction protein localisation and on the modulation of epithelial permeability under physiological conditions or under an inflammatory stress. For this purpose, we first confirmed the accumulation of PUFA in phospholipid fractions of Caco-2 cells upon 7 days of incubation with specific PUFA. Thereafter, Caco-2 cells were cultured in inserts, which provide a model of the human intestinal barrier. Accumulation of dietary n-3 PUFA in phospholipids did not affect the presence of occludin in tight junction complexes, while that of dietary n-6 PUFA decreased it. Whatever the PUFA, at 30 µM, no distortion of the Caco-2 barrier function was observed. Otherwise, 150 µM of docosahexaenoic acid (DHA) affected ZO-1 intensity under normal conditions, but not occludin or the barrier function parameters. Finally, to simulate an inflammatory state, cells were exposed for 24 h to interleukin-1ß, tumor necrosis factor-α, interferon-γ at their basolateral side and to lypopolysaccharides at both sides. DHA limited the effect of inflammatory stimulus on occludin, ZO-1 and barrier function. In conclusion, this study has evidenced the specific effect of individual PUFA to modulate occludin and ZO-1 localization, according to the inflammatory status of this in vitro model of the intestinal barrier.

Modulation of mesenchymal stem cell actin organization on conventional microcarriers for proliferation and differentiation in stirred bioreactors.

Tissue engineering applications require an appropriate combination of a cell population, biochemical factors and scaffold materials. In this field, mesenchymal stem cells (MSCs) emerge as an attractive cell population, due to their ready availability and their potential to be differentiated into various mesodermal cell types. Commercially available microcarriers have been recently recognized as an efficient tool for the propagation of such cells compared to traditional monolayer culture, enabling efficient scale-up and serving as a cell delivery system. The organization of actin as well as the induction of its effectors was previously shown to affect dramatically both proliferation and differentiation of MSCs in monolayer culture. To achieve mass scale production of differentiated cells derived from MSCs in scalable stirred bioreactors, this work aims at rationally screening microcarriers based on the characterization of actin organization. First, among the various supports tested, gelatin-based microcarriers were found to be most suitable for MSC expansion, due to their best-adapted actin organization compared to monolayer cultures. Secondly, the proper actin organization on Cultispher-S was closely linked to its ability to bind serum adhesion molecules enabling Rho GTPase activation. Finally, by modulating actin behaviour, it was feasible to efficiently guide MSC differentiation on microcarriers. Taken together, these results show that controlling actin behaviour is a good strategy toward mass scale sequential expansion followed by differentiation of MSCs in a microcarrier based bioreactor.

Selective regulation of maize plasma membrane aquaporin trafficking and activity by the SNARE SYP121.

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K(+) channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K(+) channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis.

ATM localization and gene expression in the adult mouse eye.

PURPOSE: High levels of metabolism and oxygen consumption in most adult murine ocular compartments, combined with exposure to light and ultraviolet (UV) radiation, are major sources of oxidative stress, causing DNA damage in ocular cells. Of all mammalian body cells, photoreceptor cells consume the largest amount of oxygen and generate the highest levels of oxidative damage. The accumulation of such damage throughout life is a major factor of aging tissues. Several multiprotein complexes have recently been identified as the major sensors and mediators involved in the maintenance of DNA integrity. The activity of these complexes initially seemed to be restricted to dividing cells, given their ultimate role in major cell cycle checkpoints. However, it was later established that they are also active in post-mitotic cells. Recent findings demonstrate that the DNA damage response (DDR) is essential for the development, maintenance, and normal functioning of the adult central nervous system. One major molecular factor in the DDR is the protein, ataxia telangiectasia mutated (ATM). It is required for the rapid induction of cellular responses to DNA double-strand breaks. These cytotoxic DNA lesions may be caused by oxidative damage. To understand how ATM prevents oxidative stress and participates in the maintenance of genomic integrity and cell viability of the adult retina, we determined the ATM expression patterns and studied its localization in the adult mouse eye. METHODS: Atm gene expression was analyzed by RT-PCR experiments and its localization by in situ hybridization on adult mouse ocular and cerebellar tissue sections. ATM protein expression was determined by western blot analysis of proteins homogenates extracted from several mouse tissues and its localization by immunohistochemistry experiments performed on adult mouse ocular and cerebellar tissue sections. In addition, subcellular localization was realized by confocal microscopy imaging of ocular tissue sections, with a special focus on retinal cells. RESULTS: Using RT-PCR, we detected a band of the expected size, with its sequence matching the amplified Atm cDNA sequence. Atm mRNA was detected in most cell bodies of the adult mouse eye by in situ hybridization of ocular tissue sections with specific digoxigenin-labeled PCR-amplified cDNA probes. Western blotting with different specific antibodies revealed bands corresponding to the expected sizes of ATM and its active forms (ATMp). These bands were not observed in the analysis of protein homogenates from Atm-deficient mouse tissues. ATM immunoreactivity was detected in the nucleus of all adult mice retinal cells and in most non-neuronal ocular cell types. The active phosphorylated form of ATM was also present in the retina as well as in non-neuronal cells of the adult mouse eye. However, its subcellular localization differed as a function of the cell type examined. A major finding of this study was that ATMp immunostaining in photoreceptor cells was exclusively in the cytoplasm, whereas ATM immunostaining was only in the nucleus of these cells. Furthermore, the specific and distinct ATM and ATMp immunolabeling patterns in photoreceptor cells were identical to those observed in the adult mouse cerebellar granule cells. CONCLUSIONS: We report the expression profile of Atm gene and protein in the adult mouse eye. In particular, we observed a difference between the localization patterns of the active and inactive forms of ATM in photoreceptor cells. These localization patterns suggest that ATM and its phosphorylated activated form may be involved in both the protection of cells from oxidative damage and the maintenance of ocular cell structure and function. The protection mechanisms mediated by the two forms of ATM appear to be particularly important in maintaining photoreceptor integrity.