Histochemical method for dipeptidyl aminopeptidase II with a new anthraquinonyl hydrazide substrate.

A new method for the histochemical visualization of lysosomal aminopeptidase dipeptidyl peptidase II activity (DPP II) is developed. The substrate L-Lys-L-Ala-5-chloro-1-anthraquinonylhydrazide-2HBr (Lys-Ala-CAH) is readily hydrolyzed by the enzyme to release 5-Cl-1-anthraquinonylhydrazine (CAH). The last compound is simultaneously coupled to an aromatic aldehyde, e.g. 4-nitrobenzaldehyde (p-NBA) or piperonal (3,4-methylenedioxybenzaldehyde; PPL), to form a highly insoluble deeply colored hydrazone, marking the enzyme locations. Using the new method, DPP II is successfully localyzed in tissue sections from different rat organs.     

Foam separation of DNA and proteins from solutions

Foam separation on BSA-DNA (bovine serum albumine/deoxyribonucleic acid) and Lysozyme-DNA systems is performed. The separation of the total protein from DNA is evaluated for dissociated chromatin solution. Foam separation for the same systems is done also by a new method of creating a pressure gradient in the Plateau-Gibbs borders in the foam and obtaining a "dry" foam. It is shown that the effectiveness of the foam separation can be improved significantly by the application of the latter method. Some factors (pH, initial concentration of the solution, expansion factor of the foam) influencing the separation of proteins from DNA in the foam and in the residual solution are studied as well.     

Protein-Protein Interactions in a Crowded Environment: An Analysis via Cross-Docking Simulations and Evolutionary Information

Large-scale analyses of protein-protein interactions based on coarse-grain molecular docking simulations and binding site predictions resulting from evolutionary sequence analysis, are possible and realizable on hundreds of proteins with variate structures and interfaces. We demonstrated this on the 168 proteins of the Mintseris Benchmark 2.0. On the one hand, we evaluated the quality of the interaction signal and the contribution of docking information compared to evolutionary information showing that the combination of the two improves partner identification. On the other hand, since protein interactions usually occur in crowded environments with several competing partners, we realized a thorough analysis of the interactions of proteins with true partners but also with non-partners to evaluate whether proteins in the environment, competing with the true partner, affect its identification. We found three populations of proteins: strongly competing, never competing, and interacting with different levels of strength. Populations and levels of strength are numerically characterized and provide a signature for the behavior of a protein in the crowded environment. We showed that partner identification, to some extent, does not depend on the competing partners present in the environment, that certain biochemical classes of proteins are intrinsically easier to analyze than others, and that small proteins are not more promiscuous than large ones. Our approach brings to light that the knowledge of the binding site can be used to reduce the high computational cost of docking simulations with no consequence in the quality of the results, demonstrating the possibility to apply coarse-grain docking to datasets made of thousands of proteins. Comparison with all available large-scale analyses aimed to partner predictions is realized. We release the complete decoys set issued by coarse-grain docking simulations of both true and false interacting partners, and their evolutionary sequence analysis leading to binding site predictions. Download site: http://www.lgm.upmc.fr/CCDMintseris/     

Ultra-Rapid Vision in Birds

Flying animals need to accurately detect, identify and track fast-moving objects and these behavioral requirements are likely to strongly select for abilities to resolve visual detail in time. However, evidence of highly elevated temporal acuity relative to non-flying animals has so far been confined to insects while it has been missing in birds. With behavioral experiments on three wild passerine species, blue tits, collared and pied flycatchers, we demonstrate temporal acuities of vision far exceeding predictions based on the sizes and metabolic rates of these birds. This implies a history of strong natural selection on temporal resolution. These birds can resolve alternating light-dark cycles at up to 145 Hz (average: 129, 127 and 137, respectively), which is ca. 50 Hz over the highest frequency shown in any other vertebrate. We argue that rapid vision should confer a selective advantage in many bird species that are ecologically similar to the three species examined in our study. Thus, rapid vision may be a more typical avian trait than the famously sharp vision found in birds of prey.     

Restriction of de novo pyrimidine biosynthesis inhibits Th1 cell activation and promotes Th2 cell differentiation

Leflunomide, an inhibitor of de novo pyrimidine biosynthesis, has recently been introduced as a treatment for rheumatoid arthritis in an attempt to ameliorate inflammation by inhibiting lymphocyte activation. Although the immunosuppressive ability of leflunomide has been well described in several experimental animal models, the precise effects of a limited pyrimidine supply on T cell differentiation and effector functions have not been elucidated. We investigated the impact of restricted pyrimidine biosynthesis on the activation and differentiation of CD4 T cells in vivo and in vitro. Decreased activation of memory CD4 T cells in the presence of leflunomide resulted in impaired generation and outgrowth of Th1 effectors without an alteration of Th2 cell activation. Moreover, priming of naive T cells in the presence of leflunomide promoted Th2 differentiation from uncommitted precursors in vitro and enhanced Th2 effector functions in vivo, as indicated by an increase in Ag-specific Th2 cells and in the Th2-dependent Ag-specific Ig responses (IgG1) in immunized mice. The effects of leflunomide on T cell proliferation and differentiation could be antagonized by exogenous UTP, suggesting that they were related to a profound inhibition of de novo pyrimidine biosynthesis. These results indicate that leflunomide might exert its anti-inflammatory activities in the treatment of autoimmune diseases by preventing the generation of proinflammatory Th1 effectors and promoting Th2 cell differentiation. Moreover, the results further suggest that differentiation of CD4 T cells can be regulated at the level of nucleotide biosynthesis.     

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.     

Candida albicans double-stranded DNA can participate in the host defense against disseminated candidiasis

In the present work, we studied the in vitro immunomodulatory properties of double-stranded Candida albicans DNA and its protective effect in murine disseminated candidiasis. DNA induced the production of TNF-alpha by peritoneal macrophages and splenocytes in vitro through a chloroquine-dependent mechanism. Yeast DNA acted synergistically with IFN-gamma in triggering the secretion of nitric oxide by macrophages and enabled them to stimulate the proliferation of T cells in response to soluble anti-CD3. The effect of DNA on splenocytes is associated with an enhanced synthesis of IFN-gamma, IL-2 and IL-10. In vivo, DNA decreased the mortality and lowered the kidney contamination in mice intraperitoneally inoculated with C. albicans simultaneously with an increase in the specific proliferative response and cytokine production. The present results indicate that C. albicans DNA can provide protection against disseminated infection.     

Estimation of the muscle fibre semi-length under varying joint positions on the basis of non-invasively extracted motor unit action potentials.

Changes in muscle fibre length and surface electrode position with respect to the muscle fibres affect the amplitude and frequency characteristics of surface electromyography (SEMG) in different ways. Knowledge of changes in muscle fibre length would help towards a better interpretation of the signals. The possibility of estimating the length through SEMG during voluntary contractions was checked in this study. The fibres' semi-length was estimated from the product of the conduction velocity and conduction time during which the wave of excitation propagated from the end-plate region to the ends of the fibres. Short (10 s), moderate (30% of maximum voluntary contraction) isometric contractions were performed by 10 subjects at different elbow joint angles (80-140 degrees in steps of 20 degrees ). Monopolar signals were detected non-invasively, using a two-dimensional electrode array. High spatial resolution EMG and a decomposition technique were utilised to extract single motor unit activities for triggered averaging and to estimate conduction velocity. A significant increase with joint angle was found in conduction time and estimated fibre semi-length. Changes in conduction velocity with joint angle were found to be not significant. The methodology described allows the relative changes in fibres' semi-length to be estimated from SEMG data.     

Intravenous Immunoglobulin with Enhanced Polyspecificity Improves Survival in Experimental Sepsis and Aseptic Systemic Inflammatory Response Syndromes

Sepsis is a major cause for death worldwide. Numerous interventional trials with agents neutralizing single proinflammatory mediators have failed to improve survival in sepsis and aseptic systemic inflammatory response syndromes. This failure could be explained by the widespread gene expression dysregulation known as “genomic storm” in these patients. A multifunctional polyspecific therapeutic agent might be needed to thwart the effects of this storm. Licensed pooled intravenous immunoglobulin preparations seemed to be a promising candidate, but they have also failed in their present form to prevent sepsis-related death. We report here the protective effect of a single dose of intravenous immunoglobulin preparations with additionally enhanced polyspecificity in three models of sepsis and aseptic systemic inflammation. The modification of the pooled immunoglobulin G molecules by exposure to ferrous ions resulted in their newly acquired ability to bind some proinflammatory molecules, complement components and endogenous “danger” signals. The improved survival in endotoxemia was associated with serum levels of proinflammatory cytokines, diminished complement consumption and normalization of the coagulation time. We suggest that intravenous immunoglobulin preparations with additionally enhanced polyspecificity have a clinical potential in sepsis and related systemic inflammatory syndromes.     

Nested Canalyzing Depth and Network Stability

We introduce the nested canalyzing depth of a function, which measures the extent to which it retains a nested canalyzing structure. We characterize the structure of functions with a given depth and compute the expected activities and sensitivities of the variables. This analysis quantifies how canalyzation leads to higher stability in Boolean networks. It generalizes the notion of nested canalyzing functions (NCFs), which are precisely the functions with maximum depth. NCFs have been proposed as gene regulatory network models, but their structure is frequently too restrictive and they are extremely sparse. We find that functions become decreasingly sensitive to input perturbations as the canalyzing depth increases, but exhibit rapidly diminishing returns in stability. Additionally, we show that as depth increases, the dynamics of networks using these functions quickly approach the critical regime, suggesting that real networks exhibit some degree of canalyzing depth, and that NCFs are not significantly better than functions of sufficient depth for many applications of the modeling and reverse engineering of biological networks.