Activation of platelet-derived growth factor pathway in human asthmatic pulmonary-derived mesenchymal cells

Cell cultures of mesenchymal type were obtained from biopsies taken after bronchoscopy from patients with asthma. It was possible to achieve outgrowth of fibroblast-like cells from these lung biopsies, which stained for alpha-smooth actin indicating that they were of myofibroblast type. Morphologically, two types of myofibroblasts could be observed: one intermediate form with more stretched cell shape and lamellipodia protrusions, and one more differentiated compact form of myofibroblast. The intermediate form was the most dominant type in these patients, indicating an active ongoing remodelling process. Further studies showed that platelet-derived growth factor (PDGF) might be the factor that stimulates the formation of the intermediate type of myofibroblasts, since it enhance migration of normal human lung fibroblasts 4-fold compared to control through an induced formation of stress fibers and lamellipodia protrusions. Additionally, intracellular signalling pathways involved in migration, such as RhoA and MAPkinase were stimulated 1.5-fold and 3.5-fold, respectively. By using two-dimensional (2-D) gel electrophoresis and protein identification by peptide mass finger printing matrix assisted laser desporption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS) it was possible to confirm that PDGF affected the synthesis of proteins involved in the remodelling process, such as collagen VI and post-translational forms thereof. PDGF also stimulated the production of FK506 binding protein of 65 kDa, a protein involved in smooth muscle differentiation, and proteins involved in the rearrangement of the cytoskeleton connected to migration such as the actin related protein ARP3, the T-complex protein and the heat shock protein 60. We demonstrate that PDGF has a potential pathological role in asthma and formation of subepithelial fibrosis by inducing changes in the proteome.     

Mathematical modeling of flow and kinetics in a reactor for dilute-acid hydrolysis of cellulose particles

Amathematical model to simulate the dilute-acid hydrolysis process of cellulose particles is presented. In this model, the mass is treated as a mixture of different components. A test case is considered for which transport equations for components are developed and solved together with the momentum equation for the fluid flow. To solve the model equations, a commercially available flow solver was used. All input data were taken from previously published works. For the small static mixer considered as test geometry, the result, in terms of the conversion of the cellulose particles, was reasonable. With input parameters that are relevant to a plant-size reactor, the model can be used to predict the conversion of both cellulose and hemicellulose particles.     

Synthesis and stability of monolayer-protected Au38 clusters

A synthesis strategy to obtain monodisperse hexanethiolate-protected Au38 clusters based on their resistance to etching upon exposure to a hyperexcess of thiol is reported. The reduction time in the standard Brust-Schiffrin two-phase synthesis was optimized such that Au38 were the only clusters that were fully passivated by the thiol monolayer which leaves larger particles vulnerable to etching by excess thiol. The isolated Au38 was characterized by mass spectrometry, thermogravimetric analysis, optical spectroscopy, and electrochemical techniques giving Au38(SC6)22 as the molecular formula for the cluster. These ultrasmall Au clusters behave analogously to molecules with a wide energy gap between occupied (HOMO) and unoccupied levels (LUMO) and undergo single-electron charging at room temperature in electrochemical experiments. Electrochemistry provides an elegant means to study the electronic structure and the chemical stability of the clusters at different charge states. We used cyclic voltammetry and scanning electrochemical microscopy to unequivocally demonstrate that Au38 can be reversibly oxidized to charge states z = +1 or +2; however, reduction to z = -1 leads to desorption of the protecting thiolate monolayer. Although this reductive desorption of thiol from the cluster surface is superficially analogous to electrochemical desorption of planar self-assembled monolayers (SAMs) from macroscopic electrodes, the molecular details of the process are likely to be complicated based on the current view that the thiolate monolayer in clusters is in fact composed of polymeric Au-S complexes.     

Separation and analysis of peptides and proteins from Vipera lebetina snake venom

Our studies of Levantine viper venom have demonstrated that the venom is a rich source of biomedically important proteins and peptides. The venom contains metalloproteases: thrombolytic, fibrin-degrading lebetase, an endothelial cell apoptosis inducing metalloprotease (VLAIP), factor X activator (VLFXA); serine proteases: factor V activator, bradykinin-releasing serine proteases, β-fibrinogenase, α-fibrinogenase and chymotrypsin-like protease and different other enzymes such as phosphodiesterase, 5`-nucleotidase, ribonuclease, phospholipase A2s and L-amino acid oxidase. Among nonenzymatic components venom contains: nerve growth factor, vascular endothelial growth factor, disintegrins, C-type lectins.Here we report the isolation and characterization of proteins and peptides from Vipera lebetina venom using size exclusion, ion exchange, hydrophobic interaction and affinity chromatography, HPLC, UPLC and MALDI-TOF MS methods. N-terminal sequences and internal sequences of tryptic peptides of different proteins were determined using Edman sequencing and LC-ESI-MS/MS techniques. On the basis of fragmental sequences of proteins the oligonucleotides were designed and used as primers for cDNA cloning. Using cDNA library of the venom gland of a single snake the cDNAs coding proteins were cloned and sequenced. Protein sequences were deduced from cDNA sequences.The substrate specificity of venom proteases against insulin B-chain, bradykinin, substance P, and 6-10 amino acid residues containing peptides synthesized according to potential cleavage regions of fibrinogen, factor X, factor IX, factor V, α₂-macroglobulin bait region and pregnancy zone protein were studied using MALDI-TOF mass spectrometry technique.     

LC-QTOF/MS metabolomic profiles in human plasma after a 5-week high dietary fiber intake

The objective was to investigate the alterations of plasma metabolome profiles to identify exposure and effect markers of dietary fiber intake. Subjects (n?=?25) aged 58.6 (1.1)?years (mean and SD) with a body mass index of 26.6 (0.5)?kg/m² were given a high fiber (HF) and a low fiber (LF) diet, in a 5-week randomized controlled crossover intervention. The HF diet consisted of oat bran, rye bran, and sugar beet fiber incorporated into test food products, whereas the LF diet was made of equivalent food products to the HF diet, but without adding fibers. Blood plasma samples were collected at the start and end of each intervention period and analyzed by LC-QTOF/MS. In total, 6 features in positive mode and 14 features in negative mode were significantly different between the HF and the LF diet (p?<?0.01, q?<?0.05). Two markers, 2,6-dihydroxybenzoic acid and 2-aminophenol sulfate, were increased after HF diet, along with a tentatively identified saponin derived from oat avenacosides. The untargeted metabolomics approach enabled the identification of two new markers of dietary fiber intake in human plasma. Further studies will be needed to verify if these markers could serve as compliance markers of fiber intake.     

Coupling of Gaussian Beam and Finite Difference Solvers for Semiclassical Schrödinger Equations

In the semiclassical regime, solutions to the time-dependent Schrödinger equation for molecular dynamics are highly oscillatory. The number of grid points required for resolving the oscillations may become very large even for simple model problems, making solution on a grid intractable. Asymptotic methods like Gaussian beams can resolve the oscillations with little effort and yield good approximations when the atomic nuclei are heavy and the potential is smooth. However, when the potential has variations on a small length-scale, quantum phenomena become important. Then asymptotic methods are less accurate. The two classes of methods perform well in different parameter regimes. This opens for hybrid methods, using Gaussian beams where we can and finite differences where we have to. We propose a new method for treating the coupling between the finite difference method and Gaussian beams. The new method reduces the needed amount of overlap regions considerably compared to previous methods, which improves the efficiency.     

The structure of human dermatan sulfate epimerase 1 emphasizes the importance of C5-epimerization of glucuronic acid in higher organisms

Dermatan sulfate epimerase 1 (DS-epi1, EC 5.1.3.19) catalyzes the conversion of d-glucuronic acid to l-iduronic acid on the polymer level, a key step in the biosynthesis of the glycosaminoglycan dermatan sulfate. Here, we present the first crystal structure of the catalytic domains of DS-epi1, solved at 2.4 Å resolution, as well as a model of the full-length luminal protein obtained by a combination of macromolecular crystallography and targeted cross-linking mass spectrometry. Based on docking studies and molecular dynamics simulations of the protein structure and a chondroitin substrate, we suggest a novel mechanism of DS-epi1, involving a His/double-Tyr motif. Our work uncovers detailed information about the domain architecture, active site, metal-coordinating center and pattern of N-glycosylation of the protein. Additionally, the structure of DS-epi1 reveals a high structural similarity to proteins from several families of bacterial polysaccharide lyases. DS-epi1 is of great importance in a range of diseases, and the structure provides a necessary starting point for design of active site inhibitors.     

Accuracy and precision in the determination of perfluorinated chemicals in human blood verified by interlaboratory comparisons

Perfluorinated chemicals (PFCs) are analyzed in laboratories worldwide to determine human blood levels and exposure pathways. The development of the analytical technique has been rapid in the last 10 years, and prerequisites for accurate and precise determination of PFCs in human blood at low ng/g concentrations are today readily available. The main contributing factors are the improved LC–MS instrumentations, the increased availability of native and mass labeled PFC standards, and new column materials available for chromatographic separations. The results of the first international interlaboratory study (ILS) in 2005 on PFCs revealed relatively better analytical results for human blood analyses when compared to analyses of a number of environmental matrices. The representative accuracy for the analyses of PFCs in human matrixes reported in recent years was established in the second human serum ILS in 2006. Interlaboratory standard deviations for the two human serum samples one low level concentration and one medium level concentration were found to be 12% and 16% for PFOS, respectively, and 47% and 21% for PFOA, respectively. Reported detections for all PFCs followed a frequency of PFOS > PFOA > PFHxS > PFNA > PFDA ? PFDoA ? PFDS ? PFHxA. Due to the small number of reported values for the other perfluorosulfonates and perfluorocarboxylates, standard deviations were not established.     

Deciphering the mode of action of the processive polysaccharide modifying enzyme dermatan sulfate epimerase 1 by hydrogen–deuterium exchange mass spectrometry

Distinct from template-directed biosynthesis of nucleic acids and proteins, the enzymatic synthesis of heterogeneous polysaccharides is a complex process that is difficult to study using common analytical tools. Therefore, the mode of action and processivity of those enzymes are largely unknown. Dermatan sulfate epimerase 1 (DS-epi1) is the predominant enzyme during the formation of iduronic acid residues in the glycosaminoglycan dermatan sulfate. Using recombinant DS-epi1 as a model enzyme, we describe a tandem mass spectrometry-based method to study the mode of action of polysaccharide processing enzymes. The enzyme action on the substrate was monitored by hydrogen–deuterium exchange mass spectrometry and the sequence information was then fed into mathematical models with two different assumptions of the mode of action for the enzyme: processive reducing end to non-reducing end, and processive non-reducing end to reducing end. Model data was scored by correlation to experimental data and it was found that DS-epi1 attacks its substrate on a random position, followed by a processive mode of modification towards the non-reducing end and that the substrate affinity of the enzyme is negatively affected by each additional epimerization event. It could also be shown that the smallest active substrate was the reducing end uronic acid in a tetrasaccharide and that octasaccharides and longer oligosaccharides were optimal substrates. The method of using tandem mass spectrometry to generate sequence information of the complex enzymatic products in combination with in silico modeling can be potentially applied to study the mode of action of other enzymes involved in polysaccharide biosynthesis.