Asthma, the ugly duckling of lung disease proteomics?

The human respiratory system represents a vital but vulnerable system. It is a major target for many diseases such as cancer and asthma. The incidence of these diseases has increased dramatically in the last 40-50 years. In the search for possible new therapies, many experimental tools and methods have been developed to study these diseases, ranging from animal models to in vitro studies. In the last decades, genomic and proteomic approaches have gained a lot of attention. After the major scientific breakthroughs in the field of genomics, it is now widely accepted that to understand biological processes, large-scale protein studies through proteomics techniques are required. In the battle against lung cancer, the proteomics approach has already been successfully implemented. Surprisingly, only a few proteomics studies on the ever-increasing global asthma problem have been published so far. And although proteomics also has its limitations and experimental difficulties, in our opinion, proteomics can definitely contribute to the understanding of a complex disease such as asthma. Therefore, the additional values and possibilities of proteomics in asthma research should be thoroughly investigated. A close collaboration between the different scientific disciplines may eventually lead to the development of new therapeutic strategies against asthma.     

A steady-state modeling approach to validate an in vivo mechanism of the GAL regulatory network in Saccharomyces cerevisiae.

Cellular regulation is a result of complex interactions arising from DNA-protein and protein-protein binding, autoregulation, and compartmentalization and shuttling of regulatory proteins. Experiments in molecular biology have identified these mechanisms recruited by a regulatory network. Mathematical models may be used to complement the knowledge-base provided by in vitro experimental methods. Interactions identified by in vitro experiments can lead to the hypothesis of multiple candidate models explaining the in vivo mechanism. The equilibrium dissociation constants for the various interactions and the total component concentration constitute constraints on the candidate models. In this work, we identify the most plausible in vivo network by comparing the output response to the experimental data. We demonstrate the methodology using the GAL system of Saccharomyces cerevisiae for which the steady-state analysis reveals that Gal3p neither dimerizes nor shuttles between the cytoplasm and the nucleus.     

Linear and quadratic models of point process systems: Contributions of patterned input to output

In the 1880's Volterra characterised a nonlinear system using a functional series connecting continuous input and continuous output. Norbert Wiener, in the 1940's, circumvented problems associated with the application of Volterra series to physical problems by deriving from it a new series of terms that are mutually uncorrelated with respect to Gaussian processes. Subsequently, Brillinger, in the 1970's, introduced a point-process analogue of Volterra's series connecting point-process inputs to the instantaneous rate of point-process output. We derive here a new series from this analogue in which its terms are mutually uncorrelated with respect to Poisson processes. This new series expresses how patterned input in a spike train, represented by third-order cross-cumulants, is converted into the instantaneous rate of an output point-process. Given experimental records of suitable duration, the contribution of arbitrary patterned input to an output process can, in principle, be determined. Solutions for linear and quadratic point-process models with one and two inputs and a single output are investigated. Our theoretical results are applied to isolated muscle spindle data in which the spike trains from the primary and secondary endings from the same muscle spindle are recorded in response to stimulation of one and then two static fusimotor axons in the absence and presence of a random length change imposed on the parent muscle. For a fixed mean rate of input spikes, the analysis of the experimental data makes explicit which patterns of two input spikes contribute to an output spike.     

Protein Loop Modeling Using a New Hybrid Energy Function and Its Application to Modeling in Inaccurate Structural Environments

Protein loop modeling is a tool for predicting protein local structures of particular interest, providing opportunities for applications involving protein structure prediction and de novo protein design. Until recently, the majority of loop modeling methods have been developed and tested by reconstructing loops in frameworks of experimentally resolved structures. In many practical applications, however, the protein loops to be modeled are located in inaccurate structural environments. These include loops in model structures, low-resolution experimental structures, or experimental structures of different functional forms. Accordingly, discrepancies in the accuracy of the structural environment assumed in development of the method and that in practical applications present additional challenges to modern loop modeling methods. This study demonstrates a new strategy for employing a hybrid energy function combining physics-based and knowledge-based components to help tackle this challenge. The hybrid energy function is designed to combine the strengths of each energy component, simultaneously maintaining accurate loop structure prediction in a high-resolution framework structure and tolerating minor environmental errors in low-resolution structures. A loop modeling method based on global optimization of this new energy function is tested on loop targets situated in different levels of environmental errors, ranging from experimental structures to structures perturbed in backbone as well as side chains and template-based model structures. The new method performs comparably to force field-based approaches in loop reconstruction in crystal structures and better in loop prediction in inaccurate framework structures. This result suggests that higher-accuracy predictions would be possible for a broader range of applications. The web server for this method is available at http://galaxy.seoklab.org/loop with the PS2 option for the scoring function.     

Long Fluorescence Lifetime Molecular Probes Based on Near Infrared Pyrrolopyrrole Cyanine Fluorophores for In Vivo Imaging

Fluorescence lifetime (FLT) properties of organic molecules provide a new reporting strategy for molecular imaging in the near infrared (NIR) spectral region. Unfortunately, most of the NIR fluorescent dyes have short FLT typically clustered below 1.5 ns. In this study, we demonstrate that a new class of NIR fluorescent dyes, pyrrolopyrrole cyanine dyes, have exceptionally long FLTs ranging from 3 to 4 ns, both in vitro (dimethyl sulfoxide and albumin/water solutions) and in vivo (mice). These results provide a new window for imaging molecular processes, rejecting backscattered light and autofluorescence, and multiplexing imaging information with conventional NIR fluorescent dyes that absorb and emit light at similar wavelengths.     

Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin

Actin filaments (F-actin) are protein polymers that undergo rapid assembly and disassembly and control an enormous variety of cellular processes ranging from force production to regulation of signal transduction. Consequently, imaging of F-actin has become an increasingly important goal for biologists seeking to understand how cells and tissues function. However, most of the available means for imaging F-actin in living cells suffer from one or more biological or experimental shortcomings. Here we describe fluorescent F-actin probes based on the calponin homology domain of utrophin (Utr-CH), which binds F-actin without stabilizing it in vitro. We show that these probes faithfully report the distribution of F-actin in living and fixed cells, distinguish between stable and dynamic F-actin, and have no obvious effects on processes that depend critically on the balance of actin assembly and disassembly.     

Diffusive hidden Markov model characterization of DNA looping dynamics in tethered particle experiments

In many biochemical processes, proteins bound to DNA at distant sites are brought into close proximity by loops in the underlying DNA. For example, the function of some gene-regulatory proteins depends on such 'DNA looping' interactions. We present a new technique for characterizing the kinetics of loop formation in vitro, as observed using the tethered particle method, and apply it to experimental data on looping induced by lambda repressor. Our method uses a modified ('diffusive') hidden Markov analysis that directly incorporates the Brownian motion of the observed tethered bead. We compare looping lifetimes found with our method (which we find are consistent over a range of sampling frequencies) to those obtained via the traditional threshold-crossing analysis (which can vary depending on how the raw data are filtered in the time domain). Our method does not involve any time filtering and can detect sudden changes in looping behavior. For example, we show how our method can identify transitions between long-lived, kinetically distinct states that would otherwise be difficult to discern.     

The effect of loop size in antisense and target RNAs on the efficiency of antisense RNA control.

Most natural antisense RNAs display a high degree of secondary structure with stem-loops as their most prominent feature. Mutations affecting the inhibitory activity of these RNAs most often map in or close to loop regions in both the antisense and target RNAs. The primary recognition loops often contain 5-7 unpaired nucleotides. Nucleotide changes in the loops affect the binding rate and, hence, the inhibitory effect on the activity of the target RNA. Here we address the question whether loop sizes affect binding rates between antisense and target RNAs, using the replication control system of plasmid R1 as a model system. By creating a series of loop size mutants we show that loop size alterations have strong effects on the binding rates between the two reactant RNAs in vitro, and that most of the mutations analyzed display corresponding effects on antisense RNA control in vivo. Our data suggest that the three-dimensional structures of antisense and target RNA stem-loops are crucial for determining binding rates. The implications of these results for the design of efficient artificial antisense RNA control systems are discussed.     

Defining the roles of Asn-128, Glu-129 and Phe-130 in loop A of the 5-HT3 receptor

The ligand binding pocket of Cys-loop receptors consists of a number of binding loops termed A-F. Here we examine the 5-HT3 receptor loop A residues Asn-128, Glu-129 and Phe-130 using modelling, mutagenesis, radioligand binding and functional studies on HEK 293 cells. Replacement of Asn-128 results in receptors that have wild type [3H]granisetron binding characteristics but large changes (ranging from a five-fold decrease to a 1500-fold increase) in the 5-HT EC50 when compared to wild type receptors. Phe-130 mutant receptors show both increases and decreases in Kd and EC50 values, depending on the amino acid substituted. The most critical of these residues appears to be Glu-129; its replacement with a range of other amino acids results in non-binding and non-functional receptors. Lack of binding and function in some, but not all, of these receptors is due to poor membrane expression. These data suggest that Glu-129 is important primarily for receptor expression, although it may also play a role in ligand binding; Phe-130 is important for both ligand binding and receptor function, and Asn-128 plays a larger role in receptor function than ligand binding. In light of these results, we have created two new homology models of the 5-HT3 receptor, with alternative positions of loop A. In our preferred model Glu-129 and Phe-130 contribute to the binding site, while the location of Asn-128 immediately behind the binding pocket could contribute to the conformation changes that result in receptor gating. This study provides a new model of the 5-HT3 receptor binding pocket, and also highlights the importance of experimental data to support modelling studies.     

Toward the development of generally applicable models of the microbial loop in aquatic ecosystems

Simulation modeling has been an integral, albeit ad hoc, component of the field of aquatic microbial ecology for the past two decades. One of the most critical steps in simulation modeling is the initial formulation of a clear set of questions and goals. It is doubtful that a single generic model could be constructed to address adequately all questions of interest concerning the microbial loop because of the tremendous range in time scales that define these questions. Progress in the field of aquatic microbial ecology will benefit from an integrated research program including experimental and modeling approaches. A submodel of bacterial utilization of various qualities of organic matter that we have under construction is presented. This submodel will be a component of a larger model to evaluate the effects of quality and quantity of organic matter and inorganic nutrient inputs on estuarine food web structure and efficiency. The overall model will be general enough in its structure that it should be applicable to a wide range of questions concerning the microbial loop, with time scales ranging from hours to days.     

Degradation of the D1 protein of photosystem II under illumination in vivo: two different pathways involving cleavage or intermolecular cross-linking

The D1 protein of the photosystem II reaction center turns over the most rapidly of all the proteins of the thylakoid membrane under illumination in vivo. In vitro, the D1 protein sustained cleavage in a surface-exposed loop (DE loop) or cross-linking with another reaction center protein, the D2 protein or cytochrome b(559), under illumination. We found that the D1 protein was damaged in essentially the same way in vivo, although the resultant fragments and cross-linked adducts barely accumulated due to digestion by proteases. In vitro studies detected a novel stromal protease(s) that digested the adducts but not the monomeric D1 protein. These observations suggest that, in addition to cleavage, the cross-linking reactions themselves are processes involved in complete degradation of the D1 protein in vivo. Peptide mapping experiments located the cross-linking sites with the D2 protein among residues 226-244, which includes the cross-linking site with cytochrome b(559) [Barbato, R., et al. (1995) J. Biol. Chem. 270, 24032-24037], in the N-terminal part of the DE loop, while N-terminal amino acid sequencing of the fragment located the cleavage site around residue 260 in the C-terminal part of the loop. We propose a model explaining the occurrence of simultaneous cleavage and cross-linking and discuss the mechanisms of complete degradation of the D1 protein in vivo.     

Activation of cyclophosphamide using perfused rat liver system and induction of sister chromatid exchanges in vitro

Summary Active substance(s) probably comprised alkylating intermediate(s) were obtained from cyclophosphamide through recirculation in the perfused rat liver system. The circulated output fluid induced a dose-related increase of sister chromatid exchanges on human lymphocytes with a highest score of 48,40 in vitro without the presence of a metabolic activator such as S-9mix. A new test system for the detection of in vitro inactive enviromental mutagens was discussed.     

ATP keeps exocytosis sites in a primed state but is not required for membrane fusion: an analysis with Paramecium cells in vivo and in vitro

We have tried to specify a widespread hypothesis on the requirement of ATP for exocytosis (membrane fusion). With Paramecium tetraurelia cells, synchronously (approximately 1 s) exocytosing trichocysts, ATP pools have been measured in different strains, including wild type cells, "non-discharge" (nd), "trichless" (tl), and other mutations. The occurrence of a considerable and rapid ATP consumption also in nd and tl mutations as well as its time course (with a maximum 3-5 s after exocytosis) in exocytosis-competent strains does not match the actual extent of exocytosis performance. However, from in vivo as well as from in vitro experiments, we came to the conclusion that ATP might be required to keep the system in a primed state and its removal might facilitate membrane fusion. (For the study of exocytosis in vitro we have developed a new system, consisting of isolated cortices). In vivo as well as in vitro exocytosis is inhibited by increased levels of ATP or by a nonhydrolyzable ATP analogue. In vitro exocytosis is facilitated in ATP-free media. In vivo-microinjected ATP retards exocytosis in response to chemical triggers, whereas microinjected apyrase triggers exocytosis without exogenous trigger. Experiments with this system also largely exclude any overlaps with other processes that normally accompany exocytosis. Our data also explain why it was frequently assumed that ATP would be required for exocytosis. We conclude that membrane fusion during exocytosis does not require the presence of ATP; the occurrence of membrane fusion might involve the elimination of ATP from primed fusogenic sites; most of the ATP consumption measured in the course of exocytosis may be due to other effects, probably to recovery phenomena.     

The immune status of Schwann cells: what is the role of the P2X7 receptor?

The peripheral nervous system (PNS) displays structural barriers and a lack of lymphatic drainage which strongly limit the access of molecules and cells from the immune system. In addition, the PNS has the ability to set up some specific mechanisms of immune protection to limit the pathogenicity of inflammation processes following insults by pathogens or inflammatory autoimmune diseases like the Guillain-Barré syndrome. Schwann cells are among the most prominent cells which can display immune capabilities in the PNS. Numerous in vitro studies have shown that Schwann cells were indeed able to display a large repertoire of properties, ranging from the participation to antigen presentation, to secretion of pro- and anti-inflammatory cytokines, chemokines and neurotrophic factors. In vivo studies have confirmed the immune capabilities of Schwann cells. The aim of this review is to present how Schwann cells can participate to the initiation, the regulation and the termination of the immune response in the light of the recent discovery of the Schwann cell expression of purinergic P2X7 receptors.