Autoantibodies from patients with celiac disease inhibit transglutaminase 2 binding to heparin/heparan sulfate and interfere with intestinal epithelial cell adhesion

Autoantibodies from patients with celiac disease (CD) can influence transglutaminase 2 (TG2) activity and its cellular functions, but the exact mechanisms have remained unknown. Our objective was to study whether autoantibodies could modulate TG2 binding to heparin/heparan sulfate (HS) and intestinal epithelial cell attachment to fibronectin-TG2 matrix. Anti-TG2 antibodies were purified by TG2 affinity chromatography from sera of patients with active CD. Serum and antibody effects on TG2 binding to heparin/HS, on transamidase activity of TG2, as well as on Caco-2 cell attachment to fibronectin-TG2 matrix were assessed using microplate assays. Both sera and purified anti-TG2 antibodies from CD patients with high anti-TG2 IgA levels reduced TG2 binding to heparin/HS as compared with those with low anti-TG2 IgA or controls. There was a negative correlation between anti-TG2 IgA levels and TG2 binding to heparin/HS. Treatment of fibronectin-TG2 coated wells with CD patients' sera or purified anti-TG2 antibodies reduced attachment of Caco-2 cells onto the plate as compared with the control samples. The effect of CD patients' antibodies on Caco-2 cell attachment to fibronectin-TG2 matrix occurred independently of the inhibition of cell adhesion by Arg-Gly-Asp sequence containing peptides. Anti-TG2 autoantibodies had no effect on transamidase activity of TG2 in vitro. We suggest that modulation of adhesion function of TG2 by autoantibodies from patients with CD could be related to the inhibition of TG2 binding to HS residues of cell surface proteoglycans and could have possible implications for CD pathogenesis.     

Binding of a C-end rule peptide to the neuropilin-1 receptor: a molecular modeling approach

Neuropilin-1 (NRP-1) is a receptor that plays an essential role in angiogenesis, vascular permeability, and nervous system development. Previous studies have shown that peptides with an N-terminal Arg, especially peptides with the four-residue consensus sequence R/K/XXR/K, bind to NRP-1 cell surfaces. Peptides containing such consensus sequences promote binding and internalization into cells, while blocking the C-terminal Arg (or Lys) prevents the internalization. In this study, we use molecular dynamics simulations to model the structural properties of the NRP-1 complex with a prototypic CendR peptide, RPAR. Our simulations show that RPAR binds NRP-1 through specific interactions of the RPAR C-terminus: three hydrogen bonds and a salt bridge anchor the ligand in the receptor pocket. The modeling results were used as the starting point for a systematic computational study of new RPAR analogues based on chemical modifications of their natural amino acids. Comparison of the structural properties of the new peptide-receptor complexes with the original organization suggests that some of the analogues can increase the binding affinity while reducing the natural sensitivity of RXXR to endogenous proteases.     

Calculation of photon-count number distributions via master equations

Fitting of photon-count number histograms is a way of analysis of fluorescence intensity fluctuations, a successor to fluorescence correlation spectroscopy. First versions of the theory for calculating photon-count number distributions have assumed constant emission intensity by a molecule during a counting time interval. For a long time a question has remained unanswered: to what extent is this assumption violated in experiments? Here we present a theory of photon-count number distributions that takes account of intensity fluctuations during a counting time interval. Theoretical count-number distributions are calculated via a numerical solution of Master equations (ME), which is a set of differential equations describing diffusion, singlet-triplet transitions, and photon emission. Detector afterpulsing and dead-time corrections are also included. The ME-theory is tested by fitting a series of photon-count number histograms corresponding to different lengths of the counting time interval. Compared to the first version of fluorescence intensity multiple distribution analysis theory introduced in 2000, the fit quality is significantly improved. It is discussed how a theory of photon-count number distributions, which assumes constant emission intensity during a counting time interval, may also yield a good fit quality. We argue that the spatial brightness distribution used in calculations of the fit curve is not the true spatial brightness distribution. Instead, a number of dynamic processes, which cause fluorescence intensity fluctuations, are indirectly taken into account via the profile adjustment parameters.     

Merlin links to the cAMP neuronal signaling pathway by anchoring the RIbeta subunit of protein kinase A

The cAMP-protein kinase A (PKA) pathway, important in neuronal signaling, is regulated by molecules that bind and target PKA regulatory subunits. Of four regulatory subunits, RIbeta is most abundantly expressed in brain. The RIbeta knockout mouse has defects in hippocampal synaptic plasticity, suggesting a role for RIbeta in learning and memory-related functions. Molecules that interact with or regulate RIbeta are still unknown. We identified the neurofibromatosis 2 tumor suppressor protein merlin (schwannomin), a molecule related to the ezrin-radixin-moesin family of membrane-cytoskeleton linker proteins, as a binding partner for RIbeta. Merlin and RIbeta demonstrated a similar expression pattern in central nervous system neurons and an overlapping subcellular localization in cultured hippocampal neurons and transfected cells. The proteins were coprecipitated from brain lysates by cAMP-agarose and coimmunoprecipited from cellular lysates with specific antibodies. In vitro binding studies verified that the interaction is direct. The interaction appeared to be under conformational regulation and was mediated via the alpha-helical region of merlin. Sequence comparison between merlin and known PKA anchoring proteins identified a conserved alpha-helical PKA anchoring protein motif in merlin. These results identify merlin as the first neuronal binding partner for PKA-RIbeta and suggest a novel function for merlin in connecting neuronal cytoskeleton to PKA signaling.     

The establishment of a network of European human research tissue banks

This is a report of a workshop held on the establishment of human research tissue banking which was held in Levi, Finland 21–24 March 2002.There were 21 participants from 7 European countries. This meeting was attended by representatives from academia, research tissue banks and from the Biotech and Pharmaceutical Industries. The principal aim of the workshop was to find a way to progress the recommendations from ECVAM workshop 44 (ATLA 29, 125–134,2001) and ECVAM workshop 32 (ATLA 26, 763–777, 1998). The workshop represented the first unofficial meeting of the European Network of Research Tissue Banks (ENRTB) steering group. It is expected that in the period preceding the next workshop the ENRTB steering group will co-ordinate the ethical,legislative and organisational aspects of research tissue banking. Key issues dealt with by the Levi workshop included the practical aspects of sharing expertise and experiences across the different European members. Such collaboration between research tissue banks and end users of such material seeks to ultimately enable shared access to human tissue for medical and pharmaco-toxicological research while maintaining strict adherence to differences in legal and ethical aspects related to the use of human tissue in individual countries. This revised version was published online in July 2006 with corrections to the Cover Date.     

A novel method for analyzing [Ca2+] flux kinetics in high-throughput screening

Driven by multiparameter fluorescence readouts and the analysis of kinetic responses from biological assay systems, the amount and complexity of high-throughput screening data are constantly increasing. As a consequence, the reduction of data to a simple number, reflecting a percentage activity/inhibition, is no longer an adequate approach because valuable additional information, for example, about compound-or process-induced artifacts, is lost. Time series data such as the transient calcium flux observed after activation of Gq-coupled G protein-coupled receptors (GPCRs), are especially challenging with respect to quantity of data; typically, responses are followed for several minutes. Based on measurements taken on the fluorometric imaging plate reader, the authors have introduced a mathematical model to describe the time traces of cellular calcium fluxes mediated by the activation of GPCRs. The model describes the time series using 13 parameters, reducing the amount of data by 90% while guiding the detection of compound-induced artifacts as well as the selection of compounds for further characterization.