HGF regulates tight junctions in new nontumorigenic gastric epithelial cell line

The regulation of intercellular adhesion by hepatocyte growth factor (HGF) was examined on a novel nontumorigenic gastric epithelial cell line (IMGE-5) derived from H-2Kb-tsA58 transgenic mice. IMGE-5 cells constitutively expressed cytokeratin 18 and HGF receptors. Under permissive conditions (33 degrees C + interferon-gamma), IMGE-5 cells proliferated rapidly but did not display membrane expression of adherens and tight junction proteins. Under nonpermissive conditions, their proliferation was decreased and they displayed a strong, localized membrane expression of E-cadherin/beta-catenin and occludin/ZO-1. HGF treatment largely prevented the targeting of ZO-1 to the tight junction and induced a significant decrease of the transepithelial resistance measured across a confluent IMGE-5 cell monolayer. HGF rapidly increased the tyrosine phosphorylation of ZO-1 and decreased its association with occludin in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner. PI 3-kinase was also involved in HGF-induced migration of IMGE-5 cells. Our results demonstrate that 1) HGF prevents the appearance of ZO-1 in the membrane during epithelial cell differentiation; 2) HGF causes partial relocalization of ZO-1 to the cytoplasm and nucleus and concomitantly stimulates cell dissociation and migration; and 3) IMGE-5 cells offer a useful model for the study of gastric epithelial cell differentiation.     

Effet du stress salin sur la germination et la croissance in vitro du pistachier (Pistacia vera L.)

In order to study the salinity tolerance of pistachio (Pistacia vera L.), embryos developed from mature seeds were isolated and cultured in vitro and subjected to different NaCl concentrations (0, 42.8, 85.5, 171.1 and 256.6 mM) for 30 days. The results showed that in vitro germination of embryonic axes was not affected by the salt concentration. However, the germinated embryo survival rates decreased from 100% for the control to 62.9% for the highest salt concentration (256.6 mM).In addition, the plantlet growth (length of aerial and root parts, number of leaf produced per embryo, as well as the production of total fresh and dry matter for both aerial parts and roots) showed significant differences according the various salt concentrations. To cite this article: B. Benmahioul et al., C. R. Biologies 332 (2009).     

Molecular Interplay between Endostatin, Integrins, and Heparan Sulfate

Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. α5β1 and αvβ3 integrins form stable complexes with immobilized endostatin (K_D = ∼1.8 × 10⁻⁸ m, two-state model). Two arginine residues (Arg²⁷ and Arg¹³⁹) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the αvβ3 integrin at the interface between the β-propeller domain of the αv subunit and the βA domain of the β3 subunit. In addition, we report that α5β1 and αvβ3 integrins bind to heparin/heparan sulfate. The ectodomain of the α5β1 integrin binds to haparin with high affinity (K_D = 15.5 nm). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and α5β1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.Endostatin is an endogenous inhibitor of angiogenesis that inhibits proliferation and migration of endothelial cells (1–3). This C-fragment of collagen XVIII has also been shown to inhibit 65 different tumor types and appears to down-regulate pathological angiogenesis without side effects (2). Endostatin regulates angiogenesis by complex mechanisms. It modulates embryonic vascular development by enhancing proliferation, migration, and apoptosis (4). It also has a biphasic effect on the inhibition of endothelial cell migration in vitro, and endostatin therapy reveals a U-shaped curve for antitumor activity (5, 6). Short term exposure of endothelial cells to endostatin may be proangiogenic, unlike long term exposure, which is anti-angiogenic (7). The effect of endostatin depends on its concentration and on the type of endothelial cells (8). It exerts the opposite effects on human umbilical vein endothelial cells and on endothelial cells derived from differentiated embryonic stem cells. Furthermore, two different mechanisms (heparin-dependent and heparin-independent) may exist for the anti-proliferative activity of endostatin depending on the growth factor used to induce cell proliferation (fibroblast growth factor 2 or vascular endothelial growth factor). Its anti-proliferative effect on endothelial cells stimulated by fibroblast growth factor 2 is mediated by the binding of endostatin to heparan sulfate (9), whereas endostatin inhibits vascular endothelial growth factor-induced angiogenesis independently of its ability to bind heparin and heparan sulfate (9, 10). The broad range of molecular targets of endostatin suggests that multiple signaling systems are involved in mediating its anti-angiogenic action (11), and although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanisms of action are not as fully elucidated as they are for other endogenous angiogenesis inhibitors (11).Endostatin binds with relatively low affinity to several membrane proteins including α5β1 and αvβ3 integrins (12), heparan sulfate proteoglycans (glypican-1 and -4) (13), and KDR/Flk1/vascular endothelial growth factor receptor 2 (14), but no high affinity receptor(s) has been identified so far. The identification of molecular interactions established by endostatin at the cell surface is a first step toward the understanding of the mechanisms by which endostatin regulates angiogenesis. We have previously characterized the binding of endostatin to heparan sulfate chains (9). In the present study we have focused on characterizing the interactions between endostatin, α5β1, αvβ3, and αvβ5 integrins and heparan sulfate. Although interactions between several integrins and endostatin have been studied previously in solid phase assays (12) and in cell models (12, 15, 16), no molecular data are available on the binding site of endostatin to the integrins. We found that two arginine residues of endostatin (Arg²⁷ and Arg¹³⁹) participate in binding to integrins and to heparan sulfate, suggesting that endostatin is not able to bind simultaneously to these molecules displayed at the cell surface. Furthermore, we have demonstrated that α5β1, αvβ3, and αvβ5 integrins bind to heparan sulfate. This may explain why both heparan sulfate and α5β1 integrins are required for the localization of endostatin in lipid rafts, in support of the model proposed by Wickström et al. (15).     

Physical properties of DNA components affecting the transposition efficiency of the <Emphasis Type="Italic">mariner Mos1</Emphasis> element

Previous studies have shown that the transposase and the inverted terminal repeat (ITR) of the Mos1 mariner elements are suboptimal for transposition; and that hyperactive transposases and transposon with more efficient ITR configurations can be obtained by rational molecular engineering. In an attempt to determine the extent to which this element is suboptimal for transposition, we investigate here the impact of the three main DNA components on its transposition efficiency in bacteria and in vitro. We found that combinations of natural and synthetic ITRs obtained by systematic evolution of ligands by exponential enrichment did increase the transposition rate. We observed that when untranslated terminal regions were associated with their respective natural ITRs, they acted as transposition enhancers, probably via the early transposition steps. Finally, we demonstrated that the integrity of the Mos1 inner region was essential for transposition. These findings allowed us to propose prototypes of optimized Mos1 vectors, and to define the best sequence features of their associated marker cassettes. These vector prototypes were assayed in HeLa cells, in which Mos1 vectors had so far been found to be inactive. The results obtained revealed that using these prototypes does not circumvent this problem. However, such vectors can be expected to provide new tools for the use in genome engineering in systems such as Caenorhabditis elegans in which Mos1 is very active.     

The extracellular matrix of the oleolytic biofilms of Marinobacter hydrocarbonoclasticus comprises cytoplasmic proteins and T2SS effectors that promote growth on hydrocarbons and lipids

The assimilation of the nearly water insoluble substrates hydrocarbons and lipids by bacteria entails specific adaptations such as the formation of oleolytic biofilms. The present article reports that the extracellular matrix of an oleolytic biofilm formed by Marinobacter hydrocarbonoclasticus at n‐hexadecane–water interfaces is largely composed of proteins typically cytoplasmic such as translation factors and chaperones, and a lesser amount of proteins of unknown function that are predicted extra‐cytoplasmic. Matrix proteins appear to form a structured film on hydrophobic interfaces and were found mandatory for the development of biofilms on lipids, alkanes and polystyrene. Exo‐proteins secreted through the type‐2 secretion system (T2SS) were shown to be essential for the formation of oleolytic biofilms on both alkanes and triglycerides. The T2SS effector involved in biofilm formation on triglycerides was identified as a lipase. In the case of biofilm formation on n‐hexadecane, the T2SS effector is likely involved in the mass transfer, capture or transport of alkanes. We propose that M. hydrocarbonoclasticus uses cytoplasmic proteins released by cell lysis to form a proteinaceous matrix and dedicated proteins secreted through the T2SS to act specifically in the assimilation pathways of hydrophobic substrates.     

Solution structure of NEMO zinc finger and impact of an anhidrotic ectodermal dysplasia with immunodeficiency-related point mutation

The regulatory NEMO (NF-κB essential modulator) protein has a crucial role in the canonical NF-κB signaling pathway notably involved in immune and inflammatory responses, apoptosis and oncogenesis. The regulatory domain is located in the C-terminal half of NEMO and contains a classical CCHC-type zinc finger (ZF). We have investigated the structural and functional effects of a cysteine to phenylalanine point mutation (C417F) in the ZF motif, identified in patients with anhidrotic ectodermal dysplasia with immunodeficiency. The solution structures of the wild type and mutant ZF were determined by NMR. Remarkably, the mutant adopts a global ββα fold similar to that of the wild type and retains thermodynamic stability, i.e., the ability to bind zinc with a native-like affinity, although the last zinc-chelating residue is missing. However, the mutation induces enhanced dynamics in the motif and leads to an important loss of stability. A detailed analysis of the wild type solution structure and experimental evidences led to the identification of two possible protein-binding surfaces that are largely destabilized in the mutant. This is sufficient to alter NEMO function, since functional complementation assays using NEMO-deficient pre-B and T lymphocytes show that full-length C417F pathogenic NEMO leads to a partial to strong defect in LPS, IL-1β and TNF-α-induced NF-κB activation, respectively, as compared to wild type NEMO. Altogether, these results shed light onto the role of NEMO ZF as a protein-binding motif and show that a precise structural integrity of the ZF should be preserved to lead to a functional protein-recognition motif triggering full NF-κB activation.     

Abiotic stresses increase plant regeneration ability

In disturbed habitats, vegetative regeneration is partly ruled by plant reserves and intrinsic growth rates. Under nutrient-limiting conditions, perennial plants tend to exhibit an increased allocation to storage organs. Under mechanically stressful conditions, plants also tend to increase allocation to below-ground biomass and storage organs. We tested whether those stresses acting differently on plants (nutrient level versus mechanical forces) led to similar effect on storage organs and regeneration ability. We measured, for an aquatic plant species, (1) the size and allocation to storage organs (stems) and (2) the regeneration ability of the storage organs. Plant stems were collected in 4 habitats ranked along a nutrient stress gradient, and having encountered null versus significant mechanical stress (flowing water). All stems were placed in similar neutral conditions and left for a period of 6 weeks before measuring their survival and growth. Dry mass allocation to the storage organ (stem) was higher in stressful habitats. Moreover, stress encountered by plants before the experiment significantly affected regeneration: stems of previously stressed plants (i.e. plants that had grown in nutrient-poor or mechanically stressful habitats) survived better than unstressed ones. Stems of plants having encountered mechanical stress before the experiment had increased growth in nutrient-rich habitats but reduced growth in the poorest habitats. These results demonstrate that regeneration could rely on the level of stress previously encountered by plants. Stress could lead to greater regeneration ability following mechanical failure. The possible mechanisms involved in these results are discussed.     

Field and mesocosm trials on passive sampling for the study of adsorption and desorption behaviour of lipophilic toxins with a focus on OA and DTX1

It has been demonstrated that polymeric resins can be used as receiving phase in passive samplers designed for the detection of lipophilic marine toxins at sea and was referred to as solid phase adsorption toxin tracking (SPATT). The present study describes the uptake and desorption behaviour of the lipophilic marine toxins okadaic acid (OA) and dinophysistoxin-1 (DTX1) from Prorocentrum lima cultures by five styrene—divinylbenzene based polymeric resins Sepabeads^® SP850, Sepabeads^® SP825L, Amberlite^® XAD4, Dowex^® Optipore^® L-493 and Diaion^® HP-20. All resins accumulated OA and DTX1 from the P. lima culture with differences in adsorption rate and equilibrium rate. Following statistical evaluation, HP-20, SP850 and SP825L demonstrated similar adsorption rates. However, possibly due to its larger pore size, the HP-20 did not seem to reach equilibrium within 72 h exposure as opposed to the SP850 and SP825L. This was confirmed when the resins were immersed at sea for 1 week on the West Coast of Ireland. Furthermore, this work also presents a simple and efficient extraction method suitable to SPATT samplers exposed to artificial or natural culture media.