Involvement of Membrane GRP78 in Trophoblastic Cell Fusion

Background

Glucose-regulated protein 78 (GRP78) is highly expressed in first trimester cytrophoblastic cells (CTBs), especially in syncytiotrophoblast (STB). However, the role of GRP78 in these cells has never been investigated.

Methodology/Principal Findings

In this study, we have examined the role of GRP78 in trophoblast fusion using the Bewo choriocarcinoma cell line as a model of cytotrophoblast fusion. Down regulation of GRP78 by siRNA or chemical inhibitors and use of antibodies against GRP78 in culture medium significantly decreased forskolin-induced fusion capacity of Bewo cells suggesting the involvement of membrane GRP78 in trophoblast fusion. GRP78 expression was also studied in preeclamptic (PE) CTBs which are known to have lower fusion capacity compared to control CTBs. Interestingly, despite the increase of GRP78 mRNA in PE CTBs, membrane GRP78 is significantly decreased in PE CTBs compared to control CTBs, suggesting that relocation of GRP78 from the endoplasmic reticulum to cell surface is probably altered in PE CTBs.

Conclusions

Our results imply that membrane GRP78 could play an important role in syncytialisation. They also suggest that deregulation of GRP78 expression or relocation at cell surface might be involved in pregnancy complication associated with defective syncytialisation, such as preeclampsia.     

Brassinosteroids, microtubules and cell elongation in Arabidopsis thaliana. II. Effects of brassinosteroids on microtubules and cell elongation in the bul1 mutant

In order to elucidate the involvement of brassinosteroids in the cell elongation process leading to normal plant morphology, indirect immunofluorescence and molecular techniques were use to study the expression of tubulin genes in the bul1-1 dwarf mutant of Arabidopsis thaliana (L.) Heynh., the characteristics of which are reported in this issue (M. Catterou et al., 2001). Microtubules were studied specifically in the regions of the mutant plant where the elongation zone is suppressed (hypocotyls and petioles), making the reduction in cell elongation evident. Indirect immunofluorescence of α-tubulin revealed that very few microtubules were present in mutant cells, resulting in the total lack of the parallel microtubule organization that is typical of elongating cells in the wild type. After brassinosteroid treatment, microtubules reorganized and became correctly oriented, suggesting the involvement of brassinosteroids in microtubule organization. Molecular analyses showed that the microtubule reorganization observed in brassinosteroid-treated bul1-1 plants did not result either from an activation of tubulin gene expression, or from an increase in tubulin content, suggesting that a brassinosteroid-responsive pathway exists which allows microtubule nucleation/organization and cell elongation without activation of tubulin gene expression. Received: 28 April 2000 / Accepted: 6 October 2000     

Roflumilast N-Oxide Prevents Cytokine Secretion Induced by Cigarette Smoke Combined with LPS through JAK/STAT and ERK1/2 Inhibition in Airway Epithelial Cells

Cigarette smoke is a major cause of chronic obstructive pulmonary disease (COPD). Airway epithelial cells and macrophages are the first defense cells against cigarette smoke and these cells are an important source of pro-inflammatory cytokines. These cytokines play a role in progressive airflow limitation and chronic airways inflammation. Furthermore, the chronic colonization of airways by Gram-negative bacteria, contributes to the persistent airways inflammation and progression of COPD. The current study addressed the effects of cigarette smoke along with lipolysaccharide (LPS) in airway epithelial cells as a representative in vitro model of COPD exacerbations. Furthermore, we evaluated the effects of PDE4 inhibitor, the roflumilast N-oxide (RNO), in this experimental model. A549 cells were stimulated with cigarette smoke extract (CSE) alone (0.4% to 10%) or in combination with a low concentration of LPS (0.1 µg/ml) for 2 h or 24 h for measurement of chemokine protein and mRNAs and 5–120 min for protein phosphorylation. Cells were also pre-incubated with MAP kinases inhibitors and Prostaglandin E₂ alone or combined with RNO, before the addition of CSE+LPS. Production of cytokines was determined by ELISA and protein phosphorylation by western blotting and phospho-kinase array. CSE did not induce production of IL-8/CXCL8 and Gro-α/CXCL1 from A549 cells, but increase production of CCL2/MCP-1. However the combination of LPS 0.1 µg/ml with CSE 2% or 4% induced an important production of these chemokines, that appears to be dependent of ERK1/2 and JAK/STAT pathways but did not require JNK and p38 pathways. Moreover, RNO associated with PGE₂ reduced CSE+LPS-induced cytokine release, which can happen by occur through of ERK1/2 and JAK/STAT pathways. We report here an in vitro model that can reflect what happen in airway epithelial cells in COPD exacerbation. We also showed a new pathway where CSE+LPS can induce cytokine release from A549 cells, which is reduced by RNO.     

A Single and Robust Critical Nitrogen Dilution Curve for <Emphasis Type="Italic">Miscanthus</Emphasis> × <Emphasis Type="Italic">giganteus</Emphasis> and <Emphasis Type="Italic">Miscanthus sinensis</Emphasis>

The sustainable development of miscanthus as a bioenergy feedstock requires optimizing its fertilizer inputs and, therefore, determining its nitrogen (N) requirements. The ‘critical nitrogen dilution curve’ is a powerful tool to characterize such N requirements; it relates the N concentration ([N]) in aboveground organs to their biomass, defining two domains depending on whether the N factor limits biomass growth or not. We aimed to develop such a tool in miscanthus. Using a rhizome N depletion strategy with green cutting pre-treatment over several years before the start of the experiment, we grew, in 2014, two cultivated species, Miscanthus × giganteus (M×g) and Miscanthus sinensis (Msin), at four fertilizer levels (0, 80, 160 and 240 kg N ha^?1). We found a strong nitrogen fertilization effect. The shoot [N] decreased as the aboveground biomass increased in both species and in all of the treatments. [N] was strongly correlated with leaf/stem biomass ratio. The N treatments enabled the identification of the observed critical points, i.e. points with the maximum biomass (W) and the lowest [N], on each measurement date. These points could be fitted to the following critical dilution curve that was common between M×g and Msin: N concentration (Nc) (critical [N], g N kg^?1) = 27.0 W ^?0.48 when W > 1 t ha^?1 and Nc = 27.0 when W ≤ 1. This curve was validated by literature data, separated into N-limited or not-limited conditions. The similarity of the curves between the two species was due to compensation between leaf/stem biomass ratio and [N] in the stems. This curve is helpful to diagnose the crop N status and define the optimal fertilizer requirements of miscanthus crops.     

A Highlights from MBoC Selection: Exploratory polarization facilitates mating partner selection in Saccharomyces cerevisiae

Yeast decode pheromone gradients to locate mating partners, providing a model for chemotropism. How yeast polarize toward a single partner in crowded environments is unclear. Initially, cells often polarize in unproductive directions, but then they relocate the polarity site until two partners’ polarity sites align, whereupon the cells “commit” to each other by stabilizing polarity to promote fusion. Here we address the role of the early mobile polarity sites. We found that commitment by either partner failed if just one partner was defective in generating, orienting, or stabilizing its mobile polarity sites. Mobile polarity sites were enriched for pheromone receptors and G proteins, and we suggest that such sites engage in an exploratory search of the local pheromone landscape, stabilizing only when they detect elevated pheromone levels. Mobile polarity sites were also enriched for pheromone secretion factors, and simulations suggest that only focal secretion at polarity sites would produce high pheromone concentrations at the partner’s polarity site, triggering commitment.     

Mechanisms that ensure monogamous mating in Saccharomyces cerevisiae

Haploid cells of the budding yeast Saccharomyces cerevisiae communicate using secreted pheromones and mate to form diploid zygotes. Mating is monogamous, resulting in the fusion of precisely one cell of each mating type. Monogamous mating in crowded conditions, where cells have access to more than one potential partner, raises the question of how multiple-mating outcomes are prevented. Here we identify mutants capable of mating with multiple partners, revealing the mechanisms that ensure monogamous mating. Before fusion, cells develop polarity foci oriented toward potential partners. Competition between these polarity foci within each cell leads to disassembly of all but one focus, thus favoring a single fusion event. Fusion promotes the formation of heterodimeric complexes between subunits that are uniquely expressed in each mating type. One complex shuts off haploid-specific gene expression, and the other shuts off the ability to respond to pheromone. Zygotes able to form either complex remain monogamous, but zygotes lacking both can re-mate.     

Lawsone,a 2-hydroxy-1,4-naphthoquinone from Lawsonia inermis (henna), produces mitochondrial dysfunctions and triggers mitophagy in Saccharomyces cerevisiae

Molecular Biology Reports - Lawsone is a natural naphthoquinone present in the henna leaf extract with several cytotoxic activities and used as precursor for synthesis of various pharmaceutical...