Variation and covariation among life-history traits in Rumex acetosella from a successional old-field gradient

In this study morphological variation and the potential for competition to affect biomass and seedling selection of the families of five populations of Rumex acetosella L. sampled along a successional old-field gradient have been investigated. Seeds from 25 families were submitted to four competitive regimes: no competition (one plant per pot), medium competition (two plants/ pot taking plants from the same population), high within-population competition (four individuals from the same population in a pot) and high between-population competition (four individuals from two different populations in a pot). Eight traits were analysed after 3 months of growth for variation among families within populations. A significant difference among families within the two older populations was recorded for sexual biomass and related components. High sensitivity of these traits to density was observed in all populations except the youngest, suggesting specialization to particular environmental conditions in late successional populations, and a good adaptive capacity to buffer environmental variation in the pioneer population. Little significant interaction between competitive regimes and families within populations was found, i.e. genotypes within each population showed little variation in their response to environmental variation. Genotypic variance decreased with increasing competitive conditions for the majority of the traits. However, the percentage of variance in sexual reproduction explained by family was stable among treatments. Tradeoffs between vegetative reproduction and sexual reproduction were recorded at the population level along the successional gradient, with increasing competitive conditions. As succession proceeds, we observed a decrease in sexual reproduction and an increase in vegetative reproduction. At the family level, correlation among traits were similar when plants were grown in the absence of competition and at high density, with a significant negative correlation between sexual reproduction and vegetative reproduction. For both sprout number and sexual biomass, the performance of families grown under all the treatments was positively correlated. Together these results indicate allocational constraints on the reproductive biology of R. acetosella that may be favoured by natural selection and have influenced population differentiation along the successional gradient. However, they also revealed that the potential exists for evolutionary specialization through plasticity, in response to variation in environmental conditions.     

Reproductive conflicts affect labor and immune defense in the queenless ant Diacamma sp. "nilgiri"

In many species of social Hymenoptera, totipotency of workers induces potential conflicts over reproduction. However, actual conflicts remain rare despite the existence of a high reproductive skew. One of the current hypotheses assumes that conflicts are costly and thus selected against. We studied the costs of conflicts in 20 colonies of the queenless ant Diacamma sp. "nilgiri" by testing the effects of conflicts on labor and worker immunocompetence, two parameters closely linked to the indirect fitness of workers. In this species, the dominant female is the only mated worker (gamergate) and monopolizes reproduction. We experimentally induced conflicts by splitting each colony into two groups, a control group containing the gamergate and an orphaned group displaying aggressions until a new dominant worker arises. Immunocompetence was assessed by the clearance of Escherichia coli bacteria that we injected into the ants. Time budget analysis revealed a lower rate of labor and especially brood care in orphaned groups, supporting the existence of a cost of conflicts on labor. Fifteen days after splitting, a lower immunocompetence was also found in orphaned groups, which concerned workers involved and not involved in conflicts. We propose that this immunosuppression induced by conflicts could stem from stress and not directly from aggression.     

Nitrogen metabolism responses to water deficit act through both abscisic acid (ABA)-dependent and independent pathways in Medicago truncatula during post-germination

The modulation of primary nitrogen metabolism by water deficit through ABA-dependent and ABA-independent pathways was investigated in the model legume Medicago truncatula. Growth and glutamate metabolism were followed in young seedlings growing for short periods in darkness and submitted to a moderate water deficit (simulated by polyethylene glycol; PEG) or treated with ABA. Water deficit induced an ABA accumulation, a reduction of axis length in an ABA-dependent manner, and an inhibition of water uptake/retention in an ABA-independent manner. The PEG-induced accumulation of free amino acids (AA), principally asparagine and proline, was mimicked by exogenous ABA treatment. This suggests that AA accumulation under water deficit may be an ABA-induced osmolyte accumulation contributing to osmotic adjustment. Alternatively, this accumulation could be just a consequence of a decreased nitrogen demand caused by reduced extension, which was triggered by water deficit and exogenous ABA treatment. Several enzyme activities involved in glutamate metabolism and genes encoding cytosolic glutamine synthetase (GS1b; EC 6.3.1.2.), glutamate dehydrogenase (GDH3; EC 1.4.1.1.), and asparagine synthetase (AS; EC 6.3.1.1.) were up-regulated by water deficit but not by ABA, except for a gene encoding Δ(1)-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned). Thus, ABA-dependent and ABA-independent regulatory systems would seem to exist, differentially controlling development, water content, and nitrogen metabolism under water deficit.     

Matrilin-3 switches from anti- to pro-anabolic upon integration to the extracellular matrix

The extracellular matrix (ECM) has long been viewed primarily as an organized network of solid-phase ligands for integrin receptors. During degenerative processes, such as osteoarthritis, the ECM undergoes deterioration, resulting in its remodeling and in the release of some of its components. Matrilin-3 (MATN3) is an almost cartilage specific, pericellular protein acting in the assembly of the ECM of chondrocytes. In the past, MATN3 was found required for cartilage homeostasis, but also involved in osteoarthritis-related pro-catabolic functions. Here, to better understand the pathological and physiological functions of MATN3, its concentration as a circulating protein in articular fluids of human osteoarthritic patients was determined and its functions as a recombinant protein produced in human cells were investigated with particular emphasis on the physical state under which it is presented to chondrocytes. MATN3 down-regulated cartilage extracellular matrix (ECM) synthesis and up-regulated catabolism when administered as a soluble protein. When artificially immobilized, however, MATN3 induced chondrocyte adhesion via a α5β1 integrin-dependent mechanism, AKT activation and favored survival and ECM synthesis. Furthermore, MATN3 bound directly to isolated α5β1 integrin in vitro. TGFβ1 stimulation of chondrocytes allowed integration of exogenous MATN3 into their ECM and ECM-integrated MATN3 induced AKT phosphorylation and improved ECM synthesis and accumulation. In conclusion, the integration of MATN3 to the pericellular matrix of chondrocytes critically determines the direction toward which MATN3 regulates cartilage metabolism. These data explain how MATN3 plays either beneficial or detrimental functions in cartilage and highlight the important role played by the physical state of ECM molecules.     

Induction of Therapeutic Antibodies by Vaccination against External Loops of Tumor-Associated Viral Latent Membrane Protein

Some human herpesviruses (HHV) are etiological contributors to a wide range of malignant diseases. These HHV express latent membrane proteins (LMPs), which are type III membrane proteins consistently exposed at the cell surface in these malignancies. These LMPs have relatively large cytoplasmic domains but only short extracellular loops connecting transmembrane segments that are accessible at the surface of infected cells, but they do not elicit antibodies in the course of natural infection and tumorigenesis. We report here that conformational peptides mimicking two adjacent loops of the Epstein-Barr virus (EBV) LMP1 (2LS peptides) induce high-affinity antibodies with remarkable antitumor activities in mice. In active immunization experiments, LMP1-targeting 2LS vaccine conferred tumor protection in BALB/c mice. Moreover, this tumor protection is dependent upon a humoral anti-2LS immune response as demonstrated in DO11.10 (TCR-OVA) mice challenged with LMP1-expressing tumor and in SCID mice xenografted with human EBV-positive lymphoma cells. These data provide a proof of concept for 2LS immunization against short external loops of viral LMPs. This approach might possibly be extended to other infectious agents expressing type III membrane proteins.After the primary infection, some viruses, especially human herpesviruses (HHV) such as Epstein-Barr virus (EBV), cytomegalovirus, Kaposi''s sarcoma herpesvirus (HHV8), varicella-zoster virus, and herpes simplex virus, persist lifelong in all infected individuals, most often in an asymptomatic latent form. However, in the long term, some HHV can be involved in the emergence of malignant diseases in a small subset of infected individuals. EBV-associated lymphomas and carcinomas (22, 37), HHV8-associated Kaposi''s sarcomas (30), and human cytomegalovirus-associated glioblastomas (24) are examples of beta- and gammaherpesvirus-related human malignancies. All these malignancy-associated viruses encode type III membrane proteins which are expressed during the latent state of infection and thus can be called latent membrane proteins (LMPs). These viral LMPs (vLMPs), or “multipass” membrane proteins, appeared to be necessary for virus-driven host cell survival and/or transforming activity (1, 3, 28, 31). They are regarded by some authors as evolutionary mimics of cellular chemokine/cytokine receptors, and, like cellular receptors, they recruit numerous cytoplasmic adaptors. The several transmembrane domains of these vLMPs seem to mimic activated cellular chemokine/cytokine receptor structures and to function with versatile signaling devices, reprogramming cellular signaling networks to modulate cellular function after infection. They contribute prominently to virus survival in latently infected individuals and to virus-related human pathologies, including cancer (8, 14, 19, 34, 36). Despite expressing vLMP antigens at their membrane surface, these latently infected cells are very poor in initiating effective immune responses in infected individuals, thus facilitating viral persistence in humans (2, 17, 38). One reason for this poor immunogenicity may be the constitutive cell signaling property reported for these vLMPs in latently infected cells (3, 16, 35, 38). Consequently, unnecessary overexpression and large extracellular domains for ligand binding may facilitate vLMP immune escape (3, 35, 38). Thus, a major therapeutic approach involved the discovery of naturally active compounds or pharmacological agents that specifically block viral receptor functioning (12, 35). Compounds emerged from high-throughput screening of synthetic chemical libraries, but we still lack specific agents for vLMPs, as they cross-react with cellular chemokine/cytokine receptors and cellular signaling pathways (35). Functional antibodies (Abs) recognizing membrane proteins for anticancer therapies have recently emerged, but there are very few of these and they resulted mostly from serendipity rather than from a systematic design strategy (5). To date, LMPs as a target for a virus-specific immunotherapeutic Ab strategy have not been explored extensively. Some studies have been conducted with purified full-length LMPs from EBV, a gammaherpesvirus, but these studies failed to produce or detect Abs recognizing LMP extracellular domains (10, 20, 29). One reason for this poor immunogenicity could be the too-short extracellular structure of these LMPs, which could explain the failure of latently infected individuals to produce cytolytic Abs (21). To test this hypothesis, we used as an LMP model the EBV-encoded oncoprotein LMP1 which mimics a constitutively active tumor necrosis factor receptor-like molecule and is expressed during EBV latent infection (16). This LMP1 expression was observed in most EBV-carrying malignancies (16, 22, 37), therefore causing EBV to be classified as a class I human carcinogenic agent (11). Here, we report an original humoral approach, because Abs have unlimited diversity and are often exquisitely specific and readily produced. Indeed, to overcome the too-short extracellular size of LMP, we hypothesized that synthesis of a peptide mimicking several extracellular loops of LMP would be a successful general strategy for the development of Abs with the high-pressure liquid chromatography affinity necessary for neutralizing and cytolytic effectiveness, as described previously (4, 33a). We argue here that this new process (D. Tranchand Bunel, 28 January 2003, French patent application FR0300943; D. Tranchand Bunel, 28 July 2005, U.S. Patent Office, US069140) was rewarding, as by vaccinating mice with peptides that covered two adjacent extracellular loops of LMP1 (2LS peptides), we obtained the production of neutralizing and cytolytic high-affinity Abs. Moreover, these Abs induced by 2LS peptide vaccination appeared to confer protection of mice against the development of tumors expressing LMP1.     

Traction Force Measurements of Human Aortic Smooth Muscle Cells Reveal a Motor-Clutch Behavior

The contractile behavior of smooth muscle cells (SMCs) in the aorta is an important determinant of growth, remodeling, and homeostasis. However, quantitative values of SMC basal tone have never been characterized precisely on individual SMCs. Therefore, to address this lack, we developed an in vitro technique based on Traction Force Microscopy (TFM). Aortic SMCs from a human lineage at low passages (4-7) were cultured 2 days in conditions promoting the development of their contractile apparatus and seeded on hydrogels of varying elastic modulus (1, 4, 12 and 25 kPa) with embedded fluorescent microspheres. After complete adhesion, SMCs were artificially detached from the gel by trypsin treatment. The microbeads movement was tracked and the deformation fields were processed with a mechanical model, assuming linear elasticity, isotropic material, plane strain, to extract the traction forces formerly applied by individual SMCs on the gel. Two major interesting and original observations about SMC traction forces were deduced from the obtained results: 1. they are variable but driven by cell dynamics and show an exponential distribution, with 40% to 80% of traction forces in the range 0-10 μN. 2. They depend on the substrate stiffness: the fraction of adhesion forces below 10 μN tend to decrease when the substrate stiffness increases, whereas the fraction of higher adhesion forces increases. As these two aspects of cell adhesion (variability and stiffness dependence) and the distribution of their traction forces can be predicted by the probabilistic motor-clutch model, we conclude that this model could be applied to SMCs. Further studies will consider stimulated contractility and primary culture of cells extracted from aneurysmal human aortic tissue.     

Consequences of ions and pH on the supramolecular organization of sphingomyelin and sphingomyelin/cholesterol bilayers

For drug delivery purpose the anticancer drug S12363 was loaded into ESM/Chol-liposomes using either a pH or an ammonium gradient. Association between the drug and the liposome depends markedly on the liposome membrane structure. Thus, ESM and ESM/Chol bilayer organization had been characterized by coupled DSC and XRDT as a function of both cholesterol concentration and aqueous medium composition. ESM bilayers exhibited a ripple lamellar gel phase P(beta') below the melting temperature and adopted a L(beta)-like gel phase upon Chol insertion. Supramolecular organization of ESM and ESM/Chol bilayers was not modified by citrate buffer or ammonium sulfate solution whatever the pH (3< or = pH < or =7). Nevertheless, in ESM bilayer, ammonium sulfate salt induced a peculiar organization of head groups, leading to irregular d-spacing and weakly correlated bilayers. Moreover, in the presence of salts, a weakening of van der Waals attraction forces was seen and led to a swelling of the water layer.