Allosteric Coupling in the Bacterial Adhesive Protein FimH

The protein FimH is expressed by the majority of commensal and uropathogenic strains of Escherichia coli on the tips of type 1 fimbriae and mediates adhesion via a catch bond to its ligand mannose. Crystal structures of FimH show an allosteric conformational change, but it remains unclear whether all of the observed structural differences are part of the allosteric mechanism. Here we use the protein structural analysis tool RosettaDesign combined with human insight to identify and synthesize 10 mutations in four regions that we predicted would stabilize one of the conformations of that region. The function of each variant was characterized by measuring binding to the ligand mannose, whereas the allosteric state was determined using a conformation-specific monoclonal antibody. These studies demonstrated that each region investigated was indeed part of the FimH allosteric mechanism. However, the studies strongly suggested that some regions were more tightly coupled to mannose binding and others to antibody binding. In addition, we identified many FimH variants that appear locked in the low affinity state. Knowledge of regulatory sites outside the active and effector sites as well as the ability to make FimH variants locked in the low affinity state may be crucial to the future development of novel antiadhesive and antimicrobial therapies using allosteric regulation to inhibit FimH.     

Hydrologic Variability Affects Invertebrate Grazing on Phototrophic Biofilms in Stream Microcosms

The temporal variability of streamflow is known to be a key feature structuring and controlling fluvial ecological communities and ecosystem processes. Although alterations of streamflow regime due to habitat fragmentation or other anthropogenic factors are ubiquitous, a quantitative understanding of their implications on ecosystem structure and function is far from complete. Here, by experimenting with two contrasting flow regimes in stream microcosms, we provide a novel mechanistic explanation for how fluctuating flow regimes may affect grazing of phototrophic biofilms (i.e., periphyton) by an invertebrate species (Ecdyonurus sp.). In both flow regimes light availability was manipulated as a control on autotroph biofilm productivity and grazer activity, thereby allowing the test of flow regime effects across various ratios of biofilm biomass to grazing activity. Average grazing rates were significantly enhanced under variable flow conditions and this effect was highest at intermediate light availability. Our results suggest that stochastic flow regimes, characterised by suitable fluctuations and temporal persistence, may offer increased windows of opportunity for grazing under favourable shear stress conditions. This bears important implications for the development of comprehensive schemes for water resources management and for the understanding of trophic carbon transfer in stream food webs.     

Acceleration of neuronal precursors differentiation induced by substrate nanotopography

Embryonic stem (ES) cell differentiation in specific cell lineages is a major issue in cell biology particularly in regenerative medicine. Differentiation is usually achieved by using biochemical factors and it is not clear whether mechanical properties of the substrate over which cells are grown can affect proliferation and differentiation. Therefore, we produced patterns in polydimethylsiloxane (PDMS) consisting of groove and pillar arrays of sub-micrometric lateral resolution as substrates for cell cultures. We analyzed the effect of different nanostructures on differentiation of ES-derived neuronal precursors into neuronal lineage without adding biochemical factors. Neuronal precursors adhered on PDMS more effectively than on glass coverslips. We demonstrated that neuronal yield was enhanced by increasing pillars height from 35 to 400 nm. On higher pillar neuronal differentiation reaches ～80% 96 h after plating and the largest differentiation enhancement of pillars over flat PDMS was observed during the first 6 h of culture. We conclude that PDMS nanopillars accelerate and increase neuronal differentiation.     

Revisitation of the βCl-elimination reaction of D-amino acid oxidase: new interpretation of the reaction that sparked flavoprotein dehydrogenation mechanisms

D-amino acid oxidase (DAAO) from pig has been reported to catalyze the β-elimination of Cl(-) from βCl-D-alanine via abstraction of the substrate α-H as H(+) ("carbanion mechanism") (Walsh, C. T., Schonbrunn, A., and Abeles, R. H. (1971) J. Biol. Chem. 246, 6855-6866). In view of the fundamental mechanistic importance of this reaction and of the recent reinterpretation of the DAAO dehydrogenation step as occurring via a hydride mechanism, we reinvestigated the elimination reaction using yeast DAAO. That enzyme catalyzes the same reactions as the pig enzyme but with a much higher efficiency and a substantially different kinetic behavior. The reaction is initiated by a very rapid and fully reversible dehydrogenation step. This leads to an equilibrium (k(on) ≈ k(reverse)) between the complexes of oxidized enzyme-βCl-D-alanine and reduced enzyme-βCl-iminopyruvate. In the presence of O(2) the latter complex can partition between an oxidative half-reaction and elimination of Cl(-), which proceeds at a rate of ≈50 s(-1). This step forms a complex between oxidized enzyme and enamine that is characterized by a charge transfer absorption (which describes its rates of formation and decay). A minimal scheme that lists relevant steps of the reductive and oxidative half-reactions and elimination pathways along with the estimate of the corresponding rate constants is presented. β-Elimination of Cl(-) is proposed to originate at the locus of the enzyme-βCl-iminopyruvate complex. A chemical mechanism that can account for elimination is discussed in detail.     

Kinetic and spectroscopic characterization of the putative monooxygenase domain of human MICAL-1

MICALs form a conserved multidomain protein family essential for cytoskeletal rearrangements. To complement structural information available, we produced the FAD-containing monooxygenase-like domain of human MICAL-1 (MICAL-MO) in forms differing for the presence and location of a His-tag, which only influences the protein yields. The K(m) for NADPH of the NADPH oxidase reaction is sensitive to ionic strength and type of ions. The apparent k(cat) (pH 7) is limited by enzyme reduction by NADPH, which occurs without detectable intermediates, as established by anaerobic rapid reaction experiments. The sensitivity to ionic strength and type of ions and the pH dependence of the steady-state kinetic parameters extend MICAL-MO similarity with enzymes of the p-hydroxybenzoate hydroxylase class at the functional level. The reaction is also sensitive to solvent viscosity, providing a tool to monitor the conformational changes predicted to occur during turnover. Finally, it was confirmed that MICAL-MO promotes actin depolymerization, and it was shown that F-actin, but not G-actin, stimulates NADPH oxidation by increasing k(cat) and k(cat)/K(NADPH) (≈5 and ≈200-fold, respectively) with an apparent K(m) for actin of 4.7μM, under conditions that stabilize F-actin. The time-course of NADPH oxidation shows substrate recycling, indicating the possible reversibility of MICAL effect.     

Neuroprotective properties of marrow-isolated adult multilineage-inducible cells in rat hippocampus following global cerebral ischemia are enhanced when complexed to biomimetic microcarriers

Cell-based therapies for global cerebral ischemia represent promising approaches for neuronal damage prevention and tissue repair promotion. We examined the potential of marrow-isolated adult multilineage-inducible (MIAMI) cells, a homogeneous subpopulation of immature human mesenchymal stromal cell, injected into the hippocampus to prevent neuronal damage induced by global ischemia using rat organotypic hippocampal slices exposed to oxygen-glucose deprivation and rats subjected to asphyxial cardiac arrest. We next examined the value of combining fibronectin-coated biomimetic microcarriers (FN-BMMs) with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) pre-treated MIAMI compared to EGF/bFGF pre-treated MIAMI cells alone, for their in vitro and in vivo neuroprotective capacity. Na?ve and EGF/bFGF pre-treated MIAMI cells significantly protected the Cornu Ammonis layer 1 (CA1) against ischemic death in hippocampal slices and increased CA1 survival in rats. MIAMI cells therapeutic value was significantly increased when delivering the cells complexed with FN-BMMs, probably by increasing stem cell survival and paracrine secretion of pro-survival and/or anti-inflammatory molecules as concluded from survival, differentiation and gene expression analysis. Four days after oxygen and glucose deprivation and asphyxial cardiac arrest, few transplanted cells administered alone survived in the brain whereas stem cell survival improved when injected complexed with FN-BMMs. Interestingly, a large fraction of the transplanted cells administered alone or in complexes expressed βIII-tubulin suggesting that partial neuronal transdifferentiation may be a contributing factor to the neuroprotective mechanism of MIAMI cells.     

Proteomic analysis of pork meat in the production of cooked ham

The industrial production of cooked ham from pork meat involves, as initial steps, the injection of brine and a prolonged meat massage. These processes strongly affect the quality of the final product because they determine the breakage of muscle cells and the release of their protein content. The produced dense exudates act as a glue in the final cooked ham. In order to exploit modern tools to direct the technological process, still mainly based on empirical observations and traditional recipes, we have carried out a comprehensive proteomic analysis of the exudates as a function of brine concentration, temperature, and length of meat massage. Each condition was found to generate specific protein patterns. Peptide mass fingerprinting analysis was applied allowing the identification of proteins, whose presence and/or quantity can be defined as biomarkers of meat processing, and, potentially, of final product quality.     

The actin nucleating Arp2/3 complex contributes to the formation of axonal filopodia and branches through the regulation of actin patch precursors to filopodia

The emergence of axonal filopodia is the first step in the formation of axon collateral branches. In vitro, axonal filopodia emerge from precursor cytoskeletal structures termed actin patches. However, nothing is known about the cytoskeletal dynamics of the axon leading to the formation of filopodia in the relevant tissue environment. In this study we investigated the role of the actin nucleating Arp2/3 complex in the formation of sensory axon actin patches, filopodia, and branches. By combining in ovo chicken embryo electroporation mediated gene delivery with a novel acute ex vivo spinal cord preparation, we demonstrate that actin patches form along sensory axons and give rise to filopodia in situ. Inhibition of Arp2/3 complex function in vitro and in vivo decreases the number of axonal filopodia. In vitro, Arp2/3 complex subunits and upstream regulators localize to actin patches. Analysis of the organization of actin filaments in actin patches using platinum replica electron microscopy reveals that patches consist of networks of actin filaments, and filaments in axonal filopodia exhibit an organization consistent with the Arp2/3-based convergent elongation mechanism. Nerve growth factor (NGF) promotes formation of axonal filopodia and branches through phosphoinositide 3-kinase (PI3K). Inhibition of the Arp2/3 complex impairs NGF/PI3K-induced formation of axonal actin patches, filopodia, and the formation of collateral branches. Collectively, these data reveal that the Arp2/3 complex contributes to the formation of axon collateral branches through its involvement in the formation of actin patches leading to the emergence of axonal filopodia.