Sphingosine 1-Phosphate (S1P) Receptor Agonists Mediate Pro-fibrotic Responses in Normal Human Lung Fibroblasts via S1P2 and S1P3 Receptors and Smad-independent Signaling

Synthetic sphingosine 1-phosphate receptor 1 modulators constitute a new class of drugs for the treatment of autoimmune diseases. Sphingosine 1-phosphate (S1P) signaling, however, is also involved in the development of fibrosis. Using normal human lung fibroblasts, we investigated the induction of fibrotic responses by the S1P receptor (S1PR) agonists S1P, FTY720-P, ponesimod, and SEW2871 and compared them with the responses induced by the known fibrotic mediator TGF-β1. In contrast to TGF-β1, S1PR agonists did not induce expression of the myofibroblast marker α-smooth muscle actin. However, TGF-β1, S1P, and FTY720-P caused robust stimulation of extracellular matrix (ECM) synthesis and increased pro-fibrotic marker gene expression including connective tissue growth factor. Ponesimod showed limited and SEW2871 showed no pro-fibrotic potential in these readouts. Analysis of pro-fibrotic signaling pathways showed that in contrast to TGF-β1, S1PR agonists did not activate Smad2/3 signaling but rather activated PI3K/Akt and ERK1/2 signaling to induce ECM synthesis. The strong induction of ECM synthesis by the nonselective agonists S1P and FTY720-P was due to the stimulation of S1P₂ and S1P₃ receptors, whereas the weaker induction of ECM synthesis at high concentrations of ponesimod was due to a low potency activation of S1P₃ receptors. Finally, in normal human lung fibroblast-derived myofibroblasts that were generated by TGF-β1 pretreatment, S1P and FTY720-P were effective stimulators of ECM synthesis, whereas ponesimod was inactive, because of the down-regulation of S1P₃R expression in myofibroblasts. These data demonstrate that S1PR agonists are pro-fibrotic via S1P₂R and S1P₃R stimulation using Smad-independent pathways.     

Expanding the ProteOn XPR36 biosensor into a 36-ligand array expedites protein interaction analysis

Here we demonstrate methods to expand the throughput of the ProteOn XPR36 biosensor allowing for the simultaneous kinetic characterization of several multiplexed formats, such as 36 disparate antibodies targeting the same antigen, and facilitating detailed epitope binning and mapping studies. The kinetic rate constants determined by these methods correlated with those obtained on Biacore 2000 and the absolute parameter values obtained on the ProteOn’s alginate-based GLC chip agreed closer with those from Biacore’s flat C1 chip than Biacore’s dextran-based CM4 chip. Pairwise epitope binning data from the ProteOn 36-ligand array format and those generated on an orthogonal array-based biosensor, the Octet QK384, gave similar results. In an epitope mapping study using biotinylated peptides, all three biosensor platforms were similar in their ability to identify antibodies that bound to linear epitopes. We apply alternative formats of the ProteOn array that enable a significantly higher number of assays to be conducted simultaneously than previously anticipated on this platform.     

Aging and photoperiod affect the daily rhythm pattern of atrial natriuretic peptide in the rat atrium

This study investigates the release characteristics of atrial natriuretic peptide (ANP) from young (10 weeks) and old (22 months) rat atrium. Levels of ANP release from samples of atrium were studied by organ perifusion. Rats were exposed to light:dark (LD) cycles of 12:12 or 18:6 and sacrificed at different zeitgeber time (ZT) points: ZT0, ZT6, ZT8, ZT12, ZT16, and ZT19 for LD 12:12 or ZT0, ZT9, ZT16, ZT18, ZT20, and ZT 21.5 for LD 18:6. The heart was collected, and the right atrium was removed, weighed, and perifused with Krebs-bicarbonate buffer for 100 min, including a period of 50 min for stabilization of secretion rate. ANP concentrations released by atrium did not differ between the two age groups either under LD 12:12 or under LD 18:6, except at the light:dark transition under LD 12:12 conditions where ANP levels were significantly (P < 0.05) lower in young compared to old rats. ANP exhibited daily variations in concentrations under LD 12:12, with a peak during the beginning of photophase (ZT0) in young rats and a peak at the beginning of scotophase (ZT12) in old animals. These variations were strongly modified under LD 18:6, where the pattern of the release exhibited a peak during the light phase at ZT16 in both young and old rats. This strongly suggests that the atrial ANP rhythm is dependent on the environmental light:dark cycle. Moreover, the total ANP levels released by atria in old rats were significantly increased under LD 18:6 compared to standard LD 12:12. This observation strongly suggests that old animals are more sensitive to a photoperiodic change. In conclusion, our results show that ANP concentrations in the rat atrium exhibit daily variations which are significantly affected by the daylength (photoperiod) change in aged rats.     

Biochemical and Immunocytological Characterizations of Arabidopsis Pollen Tube Cell Wall

During plant sexual reproduction, pollen germination and tube growth require development under tight spatial and temporal control for the proper delivery of the sperm cells to the ovules. Pollen tubes are fast growing tip-polarized cells able to perceive multiple guiding signals emitted by the female organ. Adhesion of pollen tubes via cell wall molecules may be part of the battery of signals. In order to study these processes, we investigated the cell wall characteristics of in vitro-grown Arabidopsis (Arabidopsis thaliana) pollen tubes using a combination of immunocytochemical and biochemical techniques. Results showed a well-defined localization of cell wall epitopes. Low esterified homogalacturonan epitopes were found mostly in the pollen tube wall back from the tip. Xyloglucan and arabinan from rhamnogalacturonan I epitopes were detected along the entire tube within the two wall layers and the outer wall layer, respectively. In contrast, highly esterified homogalacturonan and arabinogalactan protein epitopes were found associated predominantly with the tip region. Chemical analysis of the pollen tube cell wall revealed an important content of arabinosyl residues (43%) originating mostly from (1→5)-α-l-arabinan, the side chains of rhamnogalacturonan I. Finally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of endo-glucanase-sensitive xyloglucan showed mass spectra with two dominant oligosaccharides (XLXG/XXLG and XXFG), both being mono O-acetylated, and accounting for over 68% of the total ion signals. These findings demonstrate that the Arabidopsis pollen tube wall has its own characteristics compared with other cell types in the Arabidopsis sporophyte. These structural features are discussed in terms of pollen tube cell wall biosynthesis and growth dynamics.Fertilization of flowering plants requires the delivery of the two sperm cells, carried by a fast growing tip-polarized pollen tube, to the egg cell. In plants with dry stigma and solid style such as Arabidopsis (Arabidopsis thaliana), this process begins with the deposition and specific adhesion of the pollen grains on the stigmatic tissue, subsequent hydration of the pollen grains, and germination of pollen tubes (Palanivelu and Preuss, 2000). Pollen tubes invade the papillae cell wall of the stigma, enter the short style, and grow through the apoplast of the specialized transmitting tract (TT) that is filled with a nutrient-rich extracellular matrix (Kandasamy et al., 1994; Lennon et al., 1998). During this invasive growth, pollen tubes are guided to the ovules via signals that need to pass through the cell wall to reach their membrane-associated or intracellular targets (Lord and Russell, 2002; Kim et al., 2003; Boavida et al., 2005; McCormick and Yang, 2005; Johnson and Lord, 2006). In plant species with wet stigma and hollow style such as lily (Lilium longiflorum), adhesion between the pollen tube wall and the TT epidermis extracellular matrix is important for the growth of the pollen tubes toward the ovules (Mollet et al., 2000, 2007; Park et al., 2000; Chae et al., 2007). In addition to being the interface between the tube cells and the surroundings (female sporophyte or culture medium), the pollen tube wall also controls the cell shape, protects the generative cells, and allows resistance against turgor pressure (Geitmann and Steer, 2006; Geitmann, 2010).Most of our knowledge on cell wall polymers of higher plants comes from investigations on vegetative organs in which cells have diffuse growth. The cell wall is mainly composed of polysaccharides (cellulose, hemicellulose, pectin, and occasionally callose, depending on the tissue) and proteoglycans (e.g. extensin and arabinogalactan proteins [AGPs]) forming a complex network with processing enzymes.Pectins are complex wall macromolecules with uncertain supramolecular organization (Vincken et al., 2003) consisting of homogalacturonan (HG) that can be methylesterified and acetylesterified, rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and xylogalacturonan (Carpita and McCann, 2000). HG is a polymer of repeated units of (1→4)-α-d-GalUA that can be cross-linked with calcium upon block-wise action of pectin methylesterases (PMEs) on methylesterified HG (Micheli, 2001). RG-II has the same homopolymer backbone as HG but is substituted with four different oligosaccharides composed of unusual sugars, such as apiose, aceric acid, and 3-deoxy-d-manno-2-octulosonic acid, of unknown function (for review, see Caffall and Mohnen, 2009). RG-I consists of the repeating disaccharide (1→4)-α-d-GalUA-(1→2)-α-l-Rha, with a wide variety of side chains attached to the rhamnosyl residues, ranging from monomers to large oligosaccharides such as (1→4)-β-d-galactan, (1→5)-α-l-arabinan, and/or type I arabinogalactan (Caffall and Mohnen, 2009).Xyloglucan (XyG) is the major hemicellulosic polysaccharide of the primary wall of flowering plants. Classic XyG consists of a (1→4)-β-d-glucan backbone substituted with Xyl, Gal-Xyl, or Fuc-Gal-Xyl motifs, which correspond, according to the one-letter code proposed by Fry et al. (1993), to X, L, and F, respectively, G being the unsubstituted glucosyl residue of the glucan backbone. The main XyG fragments released after endo-glucanase treatment of the cell wall from wild-type Arabidopsis vegetative organs are generally XXXG, XXLG/XLXG, XXFG, and XLFG (Zablackis et al., 1995; Lerouxel et al., 2002; Nguema-Ona et al., 2006; Obel et al., 2009). In addition, O-acetylation of XyG can occur, most generally on the galactosyl residues, but its biological function is unknown (Cavalier et al., 2008). In the primary wall, XyG interacts with cellulose microfibrils via hydrogen bonds and participates in the control of cell expansion (Cosgrove, 1999).AGPs and extensin belong to the Hyp-rich glycoproteins superfamily with very high levels of type II arabinogalactan glycosylation (Nothnagel, 1997; Showalter, 2001). These proteoglycans have been implicated in many aspects of plant development, including cell expansion, cell signaling and communication, embryogenesis, wound response, and pollen tube guidance (Wu et al., 1995; Nothnagel, 1997; Seifert and Roberts, 2007; Driouich and Baskin, 2008).Despite the importance of pollen tubes for the delivery of the sperm cells to the egg, little is known about the underlying molecular mechanisms that regulate the mechanical interaction of pollen tubes with female floral tissues. There are very scarce data concerning the different components of the pollen tube cell wall. Past approaches to characterize the pollen tube cell wall are limited to a few plant genera, including Camellia (Nakamura and Suzuki, 1981), Lilium (Jauh and Lord, 1996; Mollet et al., 2002), Nicotiana (Rae et al.,1985; Li et al., 1995; Ferguson et al., 1998; Qin et al., 2007), Pinus (Derksen et al., 1999), and Zea (Rubinstein et al., 1995), and are mostly based on immunocytochemistry. These studies revealed that, depending on the species, the pollen tube cell wall contains epitopes that are found in the polymers described above, including HGs with varying levels of methylesterification, AGPs, extensin-like proteins, and low amounts of cellulose. Unlike most other plant cells, callose, a (1→3)-β-glucan, is predominant and is deposited in the wall back from the tip. Moreover, it is deposited at regular intervals to form callose plugs that maintain the tube cell in the apical expanding region of the tube and separate the viable from the degenerating region of the tube (for review, see Geitmann and Steer, 2006). Only a few reports have investigated the pollen tube of the model plant Arabidopsis. They have focused either on in vivo-grown or on in vitro-grown pollen tubes using monoclonal antibodies (MAbs) directed against a subset of cell wall epitopes present in HG, XyG, and AGPs (Lennon and Lord, 2000; Freshour et al., 2003; Pereira et al., 2006), but quantitative chemical analyses are lacking. This lack of information is most likely due to the fact that substantial amounts of pollen tube material are needed for chemical analysis, and a reproducible and efficient method for liquid culture of Arabidopsis pollen tubes had not been established until recently (Boavida and McCormick, 2007; Bou Daher et al., 2009).Here, we report the composition and localization of different cell wall polymers of in vitro-grown wild-type Arabidopsis pollen tubes based on biochemical analyses coupled to immunocytochemical investigations both at light and transmission electron microscopy (TEM) levels using recently developed MAbs. Our results show distinct patterns of labeling (tip, whole tube, and shank of the tube) depending on the recognized epitope. The most striking observations are (1) the abundance of (1→5)-α-l-arabinan in the tube wall (greater than 40 mol % of Ara), mostly localized, with LM6 and LM13, in the outer wall layer of the tube and (2) an atypical XyG matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profile with over 68% of the oligosaccharide fragments being O-acetylated.     

MAP6-F Is a Temperature Sensor That Directly Binds to and Protects Microtubules from Cold-induced Depolymerization

Microtubules are dynamic structures that present the peculiar characteristic to be ice-cold labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6/STOP family of proteins. However, the mechanism by which MAP6 stabilizes microtubules at 4 °C has not been identified. Moreover, the microtubule cold sensitivity and therefore the needs for microtubule stabilization in the wide range of temperatures between 4 and 37 °C are unknown. This is of importance as body temperatures of animals can drop during hibernation or torpor covering a large range of temperatures. Here, we show that in the absence of MAP6, microtubules in cells below 20 °C rapidly depolymerize in a temperature-dependent manner whereas they are stabilized in the presence of MAP6. We further show that in cells, MAP6-F binding to and stabilization of microtubules is temperature- dependent and very dynamic, suggesting a direct effect of the temperature on the formation of microtubule/MAP6 complex. We also demonstrate using purified proteins that MAP6-F binds directly to microtubules through its Mc domain. This binding is temperature-dependent and coincides with progressive conformational changes of the Mc domain as revealed by circular dichroism. Thus, MAP6 might serve as a temperature sensor adapting its conformation according to the temperature to maintain the cellular microtubule network in organisms exposed to temperature decrease.     

Is Diet Flexibility an Adaptive Life Trait for Relictual and Peri-Urban Populations of the Endangered Primate Macaca sylvanus?

Habitat loss, fragmentation and urban expansion may drive some species to marginal habitats while others succeed in exploiting urban areas. Species that show dietary flexibility are more able to take advantage of human activities to supplement their diet with anthropogenically abundant and accessible resources. The Barbary macaque (Macaca sylvanus) is an endangered species due to the loss of its habitat, and human pressure. The population of Gouraya National Park (Algeria) lives in a relictual habitat that constitutes about 0.6% of the species range. In addition, this population is a unique case where urban expansion favours contact zones between Barbary macaque habitats and a big city (Bejaia). We quantified the dietary composition of Gouraya macaques over an annual cycle with the objective to understand how diet flexibility of this species may help it adapt to a relictual habitat or cope with urban expansion. We recorded the phenology of plant species every month. This study shows that Gouraya macaques, compared to those living in other forest types of the distribution area, are under lower seasonal constraints. They consume a greater amount of fruit and seeds that are available throughout much of the year, and a lesser amount of costly to find and extract subterranean foods. Therefore the Gouraya relictual habitat appears as a favourable environment compared to other major habitats of that species. This study also shows that colonizing peri-urban zones increases the availability and species richness of diet resources for Barbary macaques as they consume more human foods and exotic plants than in farther sites. Adult males eat more human foods than adult females and immatures do. The exploitation of high-energy anthropogenic food could favour macaque population growth and expansion towards the city center associated with human/macaque conflicts. We recommend applying management actions to restore macaques back to their natural habitat.