Cell dynamic adhesion and elastic properties probed with cylindrical atomic force microscopy cantilever tips

Cell adhesion is required for essential biological functions such as migration, tissue formation and wound healing, and it is mediated by individual molecules that bind specifically to ligands on other cells or on the extracellular matrix. Atomic force microscopy (AFM) has been successfully used to measure cell adhesion at both single molecule and whole cell levels. However, the measurement of inherent cell adhesion properties requires a constant cell-probe contact area during indentation, a requirement which is not fulfilled in common pyramidal or spherical AFM tips. We developed a procedure using focused ion beam (FIB) technology by which we modified silicon pyramidal AFM cantilever tips to obtain flat-ended cylindrical tips with a constant and known area of contact. The tips were validated on elastic gels and living cells. Cylindrical tips showed a fairly linear force-indentation behaviour on both gels and cells for indentations >200 nm. Cylindrical tips coated with ligands were used to quantify inherent dynamic cell adhesion and elastic properties. Force, work of adhesion and elasticity showed a marked dynamic response. In contrast, the deformation applied to the cells before rupture was fairly constant within the probed dynamic range. Taken together, these results suggest that the dynamic adhesion strength is counterbalanced by the dynamic elastic response to keep a constant cell deformation regardless of the applied pulling rate.     

Comparing nutritional value and yield as functional units in the environmental assessment of horticultural production with organic or mineral fertilization

Background, aim, and scope  

We report the environmental assessment of the cultivation cycle of cauliflower (Brassica oleracea L. var. botrytis), chosen due to its high levels of natural bioactive compounds, using different fertilization practices. The functional units used during the impact assessment were linked with the quantity produced, considering different units of commercialization, or with the cauliflower quality, considering its antioxidant compounds content. Although nutrient content has been described and used as a possible functional unit, using antioxidant compounds as a functional unit has not previously been published.     

Dissecting the conserved NPxxY motif of the M3 muscarinic acetylcholine receptor: critical role of Asp-7.49 for receptor signaling and multiprotein complex formation

Acetylcholine challenge produces M(3) muscarinic acetylcholine receptor activation and accessory/scaffold proteins recruitment into a signalsome complex. The dynamics of such a complex is not well understood but a conserved NPxxY motif located within transmembrane 7 and juxtamembrane helix 8 of the receptor was found to modulate G protein activation. Here by means of receptor mutagenesis we unravel the role of the conserved M(3) muscarinic acetylcholine receptor NPxxY motif on ligand binding, signaling and multiprotein complex formation. Interestingly, while a N7.49D receptor mutant showed normal ligand binding properties a N7.49A mutant had reduced antagonist binding and increased affinity for carbachol. Also, besides this last mutant was able to physically couple to Gα(q/11) after carbachol challenge it was neither capable to activate phospholipase C nor phospholipase D. On the other hand, we demonstrated that the Asn-7.49 is important for the interaction between M(3)R and ARF1 and also for the formation of the ARF/Rho/β γ signaling complex, a complex that might determine the rapid activation and desensitization of PLD. Overall, these results indicate that the NPxxY motif of the M(3) muscarinic acetylcholine receptor acts as key conformational switch for receptor signaling and multiprotein complex formation.     

Complementation and regulatory interaction between two cloned fimbrial gene clusters of Escherichia coli strain KS71

Summary Complementation experiments with cloned DNA fragments encoding either the KS71A, the KS71B or the KS71C fimbriae of the pyelonephritogenic Escherichia coli strain KS71 were used to localise the P-fimbrillin genes and to demonstrate regulatory interactions between the cloned genes. The structural genes of the KS71A and KS71B fimbriae were located within a common 1.1 kilobase pair ClaI-SmaI fragment, and it was shown that the gene clusters for these fimbriae could complement each other in trans. The gene cluster encoding the KS71C fimbriae did not complement for the other KS71 fimbriae. A DNA fragment, located near the KS71A fimbrillin gene, was found to enhance the production of the KS71B fimbriae in trans.     

Identification of a Vitis vinifera endo‐β‐1,3‐glucanase with antimicrobial activity against Plasmopara viticola

Inducible plant defences against pathogens are stimulated by infections and comprise several classes of pathogenesis‐related (PR) proteins. Endo‐β‐1,3‐glucanases (EGases) belong to the PR‐2 class and their expression is induced by many pathogenic fungi and oomycetes, suggesting that EGases play a role in the hydrolysis of pathogen cell walls. However, reports of a direct effect of EGases on cell walls of plant pathogens are scarce. Here, we characterized three EGases from Vitis vinifera whose expression is induced during infection by Plasmopara viticola, the causal agent of downy mildew. Recombinant proteins were expressed in Escherichia coli. The enzymatic characteristics of these three enzymes were measured in vitro and in planta. A functional assay performed in vitro on germinated P. viticola spores revealed a strong anti‐P. viticola activity for EGase3, which strikingly was that with the lowest in vitro catalytic efficiency. To our knowledge, this work shows, for the first time, the direct effect against downy mildew of EGases of the PR‐2 family from Vitis.     

Multiple models for Rosaceae genomics

The plant family Rosaceae consists of over 100 genera and 3,000 species that include many important fruit, nut, ornamental, and wood crops. Members of this family provide high-value nutritional foods and contribute desirable aesthetic and industrial products. Most rosaceous crops have been enhanced by human intervention through sexual hybridization, asexual propagation, and genetic improvement since ancient times, 4,000 to 5,000 B.C. Modern breeding programs have contributed to the selection and release of numerous cultivars having significant economic impact on the U.S. and world markets. In recent years, the Rosaceae community, both in the United States and internationally, has benefited from newfound organization and collaboration that have hastened progress in developing genetic and genomic resources for representative crops such as apple (Malus spp.), peach (Prunus spp.), and strawberry (Fragaria spp.). These resources, including expressed sequence tags, bacterial artificial chromosome libraries, physical and genetic maps, and molecular markers, combined with genetic transformation protocols and bioinformatics tools, have rendered various rosaceous crops highly amenable to comparative and functional genomics studies. This report serves as a synopsis of the resources and initiatives of the Rosaceae community, recent developments in Rosaceae genomics, and plans to apply newly accumulated knowledge and resources toward breeding and crop improvement.     

Prevention of diabetes by manipulation of anti-IGRP autoimmunity: high efficiency of a low-affinity peptide

Antigen therapy may hold great promise for the prevention of autoimmunity; however, most clinical trials have failed, suggesting that the principles guiding the choice of treatment remain ill defined. Here, we examine the antidiabetogenic properties of altered peptide ligands of CD8+ T cells recognizing an epitope of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP206-214), a prevalent population of autoreactive T cells in autoimmune diabetes. We show that islet-associated CD8+ T cells in nonobese diabetic mice recognize numerous IGRP epitopes, and that these cells have a role in the outcome of protocols designed to induce IGRP206-214-specific tolerance. Ligands targeting IGRP206-214-reactive T cells prevented disease, but only at doses that spared low-avidity clonotypes. Notably, near complete depletion of the IGRP206-214-reactive T-cell pool enhanced the recruitment of subdominant specificities and did not blunt diabetogenesis. Thus, peptide therapy in autoimmunity is most effective under conditions that foster occupation of the target organ lymphocyte niche by nonpathogenic, low-avidity clonotypes.     

Light-driven activation of beta 2-adrenergic receptor signaling by a chimeric rhodopsin containing the beta 2-adrenergic receptor cytoplasmic loops

Structure-function studies of rhodopsin indicate that both intradiscal and transmembrane (TM) domains are required for retinal binding and subsequent light-induced structural changes in the cytoplasmic domain. Further, a hypothesis involving a common mechanism for activation of G-protein-coupled receptor (GPCR) has been proposed. To test this hypothesis, chimeric receptors were required in which the cytoplasmic domains of rhodopsin were replaced with those of the beta(2)-adrenergic receptor (beta(2)-AR). Their preparation required identification of the boundaries between the TM domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR necessary for formation of the rhodopsin chromophore and its activation by light and subsequent optimal activation of beta(2)-AR signaling. Chimeric receptors were constructed in which the cytoplasmic loops of rhodopsin were replaced one at a time and in combination. In these replacements, size of the third cytoplasmic (EF) loop critically determined the extent of chromophore formation, its stability, and subsequent signal transduction specificity. All the EF loop replacements showed significant decreases in transducin activation, while only minor effects were observed by replacements of the CD and AB loops. Light-dependent activation of beta(2)-AR leading to Galphas signaling was observed only for the EF2 chimera, and its activation was further enhanced by replacements of the other loops. The results demonstrate coupling between light-induced conformational changes occurring in the transmembrane domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR.