Histochemical detection of GM1 ganglioside using cholera toxin-B subunit. Evaluation of critical factors optimal for in situ detection with special emphasis to acetone pre-extraction

A comparison of histochemical detection of GM1 ganglioside in cryostat sections using cholera toxin B-subunit after fixation with 4% formaldehyde and dry acetone gave tissue-dependent results. In the liver no pre-treatment showed detectable differences related to GM1 reaction products, while studies in the brain showed the superiority of acetone pre-extraction (followed by formaldehyde), which yielded sharper images compared with the diffuse, blurred staining pattern associated with formaldehyde. Therefore, the aim of our study was to define the optimal conditions for the GM1 detection using cholera toxin B-subunit

Mutual influence of selenium nanoparticles and FGF2-STAB® on biocompatible properties of collagen/chitosan 3D scaffolds : in vitro and ex ovo evaluation

In a biological system, nanoparticles (NPs) may interact with biomolecules. Specifically, the adsorption of proteins on the nanoparticle surface may influence both the nanoparticles' and proteins' overall bio-reactivity. Nevertheless, our knowledge of the biocompatibility and risk of exposure to nanomaterials is limited. Here, in vitro and ex ovo biocompatibility of naturally based crosslinked freeze-dried 3D porous collagen/chitosan scaffolds, modified with thermostable fibroblast growth factor 2 (FGF2-STAB®), to enhance healing and selenium nanoparticles (SeNPs) to provide antibacterial activity, were evaluated. Biocompatibility and cytotoxicity were tested in vitro using normal human dermal fibroblasts (NHDF) with scaffolds and SeNPs and FGF2-STAB® solutions. Metabolic activity assays indicated an antagonistic effect of SeNPs and FGF2-STAB® at high concentrations of SeNPs. The half-maximal inhibitory concentration (IC50) of SeNPs for NHDF was 18.9 µg/ml and IC80 was 5.6 µg/ml. The angiogenic properties of the scaffolds were monitored ex ovo using a chick chorioallantoic membrane (CAM) assay and the cytotoxicity of SeNPs over IC80 value was confirmed. Furthermore, the positive effect of FGF2-STAB® at very low concentrations (0.01 µg/ml) on NHDF metabolic activity was observed. Based on detailed in vitro testing, the optimal concentrations of additives in the scaffolds were determined, specifically 1 µg/ml of FGF2-STAB® and 1 µg/ml of SeNPs. The scaffolds were further subjected to antimicrobial tests, where an increase in selenium concentration in the collagen/chitosan scaffolds increased the antibacterial activity. This work highlights the antimicrobial ability and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® and SeNPs. Moreover, we suggest that these sponges could be used as scaffolds for growing cells in systems with low mechanical loading in tissue engineering, especially in dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration. Due to their antimicrobial properties, these scaffolds are also highly promising for tissue replacement requiring the prevention of infection

Healing and angiogenic properties of collagen/chitosan scaffolds enriched with hyperstable FGF2-STAB® protein: in vitro, ex ovo and in vivo comprehensive evaluation

Wound healing is a process regulated by a complex interaction of multiple growth factors including fibroblast growth factor 2 (FGF2). Although FGF2 appears in several tissue engineered studies, its applications are limited due to its low stability both in vitro and in vivo. Here, this shortcoming is overcome by a unique nine-point mutant of the low molecular weight isoform FGF2 retaining full biological activity even after twenty days at 37 °C. Crosslinked freeze-dried 3D porous collagen/chitosan scaffolds enriched with this hyper stable recombinant human protein named FGF2-STAB® were tested for in vitro biocompatibility and cytotoxicity using murine 3T3-A31 fibroblasts, for angiogenic potential using an ex ovo chick chorioallantoic membrane assay and for wound healing in vivo with 3-month old white New Zealand rabbits. Metabolic activity assays indicated the positive effect of FGF2-STAB® already at very low concentrations (0.01 µg/mL). The angiogenic properties examined ex ovo showed enhanced vascularization of the tested scaffolds. Histological evaluation and gene expression analysis by RT-qPCR proved newly formed granulation tissue at the place of a previous skin defect without significant inflammation infiltration in vivo. This work highlights the safety and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® protein. Moreover, these sponges could be used as scaffolds for growing cells for dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration

Single and multiple dose pharmacokinetics of maritime pine bark extract (Pycnogenol) after oral administration to healthy volunteers

BACKGROUND: Since plant extracts are increasingly used as phytotherapeutics or dietary supplements information on bioavailability, bioefficacy and safety are warranted. We elucidated the plasma kinetics of genuine extract components and metabolites after single and multiple ingestion of the standardized maritime pine bark extract Pycnogenol (USP quality) by human volunteers. METHODS: Eleven volunteers received a single dose of 300 mg pine bark extract, five volunteers ingested 200 mg daily for five days to reach steady state concentrations. Plasma samples were obtained before and at defined time points after intake of the extract. Samples were analyzed by HPLC with ion-pair reagents and simultaneous UV and electrochemical detection. RESULTS: We quantified total plasma concentrations of catechin, caffeic acid, ferulic acid, taxifolin and the metabolite M1 (δ-(3,4-dihydroxy-phenyl)-γ-valerolactone). Additionally, we describe plasma time courses and steady state appearance of ten so far unknown compounds, U1 to U10. After single ingestion, compounds derived from the extract were rapidly absorbed and the majority of them were detectable over whole experimental period of 14 h. The analysis of steady state plasma samples revealed significant phase II metabolism. CONCLUSION: We present the first systematic pharmacokinetic analysis of compounds derived from maritime pine bark extract. Beyond the known constituents and metabolites we uncovered the plasma time courses of ten unknown compounds. In concert with our previous detection of anti-inflammatory bioefficacy of these plasma samples ex vivo we suggest that constituents and metabolites of Pycnogenol bear potential for disclosure of novel active principles

Observation of electron transfer mediated decay in aqueous solution

Photoionization is at the heart of X ray photoelectron spectroscopy XPS , which gives access to important information on a sample s local chemical environment. Local and non local electronic decay after photoionization in which the refilling of core holes results in electron emission from either the initially ionized species or a neighbour, respectively have been well studied. However, electron transfer mediated decay ETMD , which involves the refilling of a core hole by an electron from a neighbouring species, has not yet been observed in condensed phase. Here we report the experimental observation of ETMD in an aqueous LiCl solution by detecting characteristic secondary low energy electrons using liquid microjet soft XPS. Experimental results are interpreted using molecular dynamics and high level ab initio calculations. We show that both solvent molecules and counterions participate in the ETMD processes, and different ion associations have distinctive spectral fingerprints. Furthermore, ETMD spectra are sensitive to coordination numbers, ion solvent distances and solvent arrangemen

Protein concentration and composition in wheat flour mill streams

Protein concentration and composition varies in different part of the wheat grain and also in different mill streams. This investigation aimed at determining protein concentration, composition and disulfide crosslinkings in different wheat flour mill streams. Flour mill streams were obtained by milling grains of two Slovakian bread wheat cultivars in a Brabender Quadromat Senior test mill. Protein concentration, amount of protein groups, amount and size distribution of polymeric and monomeric proteins, free thiol groups and disulfide crosslinkings were measured. The results showed that the break flour contained high amounts of gliadins and glutenin subunits, SDS-extractable large polymeric proteins and disulfide crosslinkings. The shorts contained high amounts of SDS-extractable and SDS-unextractable smaller monomeric proteins, SDS-unextractable larger monomeric proteins and free thiol groups. The bran contained high protein concentration, high amounts of high-molecular-weight glutenin subunits, SDS-extractable and SDS-unextractable smaller monomeric proteins and SDS-unextractable larger monomeric proteins

Carcinoembryonic antigen-related cell adhesion molecule 1 is the 85-kilodalton pronase-resistant biliary glycoprotein in the cholesterol crystallization promoting low density protein-lipid complex

A pronase resistant 85-kd glycoprotein in the Concanavalin A-binding fraction (CABF) of biliary glycoproteins has been reported to act as a promotor of cholesterol crystallization. De Bruijn et al. (Gastroenterology 1996;110:1936-1944) found this protein in a low-density protein-lipid complex (LDP) with potent cholesterol crystallization promoting activity. This study identifies and characterizes this protein. An LDP was prepared from CABF by discontinuous gradient ultracentrifugation. Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotting and immunochemical staining with anti-carcinoembryonic antigen, CEA-related adhesion molecule 1 (CEACAM1) cross-reacting antibodies. Biliary concentrations of CEA cross-reacting proteins were determined in patients with and without gallstones. Two isoforms of CEACAM1 (85- and 115-kd bands), CEA and 2 CEA cross-reacting protein bands of 40 and 50 kd were found in human bile. All bands were also present in CABF, but only a subfraction of the 85-kd band found in the LDP was resistant to digestion with pronase. CEACAM1-85 exhibited potent cholesterol crystallization promoting activity in vitro and accounted for most of the activity in CABF. Total CEA cross-reacting protein concentrations were the same in gallbladder biles from patients with cholesterol and pigment gallstones but only half of those in biles from nongallstone subjects. In conclusion, we have identified the protein component of the cholesterol crystallization promoting LDP to be CEACAM1-8