Immobilization of protein to surface-grafted PEO/PPO block copolymers

Adsorption and covalent immobilization of Ig G to a grafted tetrabranched PEO/PPO block copolymer have been studied and related to the temperature-dependent properties of the grafted polymer. The investigation was performed by means of in situ ellipsometry, as well as by ESCA and ELISA measurements. The results show that the copolymer grafted to polystyrene (PS) surface contracts substantially upon increasing the temperature. A close interrelation was found between the properties of the grafted layer and the amount of protein (Ig G) that could be either adsorbed or covalently immobilized to the modified PS surface. By utilizing the reversed temperature phase behavior exhibited by these copolymers a relatively high loading of protein was obtained at temperatures close to the cloud point. By lowering the temperature after immobilization, the grafted layer regains its hydrophilicity and protein-rejecting properties. Thus, problems associated with interaction between bound protein and the underlying solid surface are minimized.     

Colorimetrische pH-Bestimmung

Ohne Zusammenfassung     

Highly diastereoselective hydrogenations leading to beta-hydroxy delta-lactones in hydroxy-protected form. A modified view of delta-lactone conformations

Enol MEM ethers 4 and 15 and the corresponding enol acetates were hydrogenated over Pd/C with very high (>99%) diastereoselectivity to saturated delta-lactones. A stereochemical generalization can be formulated thus: trans-5,6-disubstituted 1-oxa-3-cyclohexen-2-ones (e.g. 14 and 15) are hydrogenated over Pd with high selectivity from the side trans to the C(6)-substituent. A mechanistic rationalization of the stereochemical outcome in the Pd-catalyzed hydrogenation of this as well as other types of substituted alpha,beta-unsaturated delta-lactones is presented. An analysis of X-ray crystallographic data for 67 compounds demonstrated a great conformational diversity of the saturated delta-lactone ring. Besides, ab initio calculations (HF/6-31G) indicated a very high conformational mobility. Thus, the lowest calculated transition state for the conversion of the half-chair, most stable, conformer of delta-valerolactone to the boat-type conformer lies only 1.93 kcal/mol above the former. Beside these two conformers, also chair, envelope and skew conformations are accessible; all lie less than 2 kcal/mol above the half-chair. The previous conformational paradigm comprising only boat and half-chair types is incomplete.     

Interactions between a lipase and charged surfactants--a comparison between bulk and interfaces

The interaction between a charged surfactant and a lipase has been investigated by several methods. Interactions in aqueous bulk phase was studied by NMR and by microcalorimetry. Surface tension and neutron reflectivity were used for studies at the air-water interface. Interactions at the interface between a hydrophobic solid surface and water was investigated by ellipsometry. The results obtained are as follows. The cationic surfactant, tetradecyltrimethylammonium bromide (iodide in the NMR experiments), showed strong interaction at the air-water and the hydrophobic solid-water interfaces but no clear indication of an interaction in bulk phase was seen. The anionic surfactant showed no interaction with the lipase neither at the interfaces, nor in bulk. The difference in behavior of the system cationic surfactant-lipase in bulk and at the interfaces may be due to the change in enzyme conformation that is known to occur at interfaces between water and an apolar phase.     

Toward the spectrum of free polyethylene: linear alkanes studied by carbon 1s photoelectron spectroscopy and theory

Trends in carbon 1s ionization energies for the linear alkanes have been investigated using third-generation synchrotron radiation. The study comprises CH(4), C(2)H(6), C(3)H(8), C(4)H(10), C(5)H(12), C(6)H(14), and C(8)H(18). Both inter- and intramolecular shifts in ionization energy have been determined from gas-phase spectra and ab initio calculations. The shifts are decomposed into initial-state and final-state contributions and are shown to relate to the fundamental chemical properties of group electronegativity and polarizability. By extrapolation, we predict C1s spectra of larger n-alkanes, converging toward isolated strands of polyethylene.     

Mixed micellar systems of cleavable surfactants

Previous studies have shown that the alkaline hydrolysis of cleavable ester surfactants is strongly affected by aggregation. The alkaline hydrolysis of the cationic species decyl betainate (DB) is strongly enhanced by micellization, whereas the nonionic species tetra(ethylene glycol)mono-n-octanoate (TEO) is virtually protected when residing in aggregates. In the present work, mixtures of DB and TEO were studied at concentrations above the critical micelle concentration, and the rate of hydrolysis of each surfactant in the presence of the other was assessed. The micellar interaction parameter (beta) was determined from the critical micelle concentrations of various mixtures of the two surfactants. The result (beta = -2.4) indicates a moderate net attraction. The hydrolysis of the surfactants was monitored using 1H NMR. It was shown that the hydrolysis of DB exhibits the main characteristics of the pseudophase ion-exchange model and that the reaction rate decreases with an increasing molar ratio of TEO. There are indications that the hydrolysis rate parallels the expected total counterion binding to the mixed micelles. The hydrolysis of TEO was not affected by the presence of DB. However, complementary experiments showed that it is possible to accelerate or retard the hydrolysis of TEO by coaggregation with stable cationic or anionic surfactants, respectively.     

Truncated fragments in polymerase chain reaction-based DNA sequencing

Reconstitution experiments were performed by using an ordinary dye-primer protocol of a template spiked with known amounts of truncated fragments. We observed that as little as 0.2 mole-percentage of the truncated fragment caused sequence interpretation problems. Two protocols were developed for sequencing with dye-labeled terminators; this eliminates the problems with truncated fragments, which are adapted to a one-dye chemistry. One was designed for single extension sequencing using T7 DNA polymerase and one for cycle sequencing. To avoid precipitation and centrifugation and to facilitate automation, the dye-terminator protocols included the use of a biotinylated sequencing primer. Thus, the Sanger fragments were recovered and, by magnetic separation, washed and released by formamide, EDTA, and heat treatment before loading on the electrophoresis gel. Integrated procedures for sequencing PCR products using one-dye-labeled terminators suitable for automation are described. High quality data in terms of long reads and detection of polymorphisms is obtained. The protocols serve as attractive alternatives to internal labeling and dye-primer approaches.     

Solvent effect of alcohols at the L-edge of iron in solution: X-ray absorption and multiplet calculations

The local electronic structure of Fe(III) and Fe(II) ions in different alcohol solutions (methanol, ethanol, propan-1-ol) is investigated by means of soft X-ray absorption spectroscopy at the iron L 2,3-edge. The experimental spectra are compared with ligand field multiplet simulations. The solvated Fe(III) complex is found to exhibit octahedral symmetry, while a tetragonal symmetry is observed for Fe(II). A decrease in the solvent polarity increases the charge transfer from the oxygen of the alcohol to the iron ions. This conclusion is supported by Hartree-Fock calculations of the Mulliken charge distribution on the alcohols. A larger charge transfer is further observed from the solvent to Fe(III) compared to Fe(II), which is connected to the higher positive charge state of the former. Finally, iron ions in solution are found to prefer the high-spin configuration irrespective of their oxidation state.     

Complexes of Graphs with Bounded Matching Size

For positive integers k,n, we investigate the simplicial complex of all graphs G on vertex set [n] such that every matching in G has size less than k. This complex (along with other associated cell complexes) is found to be homotopy equivalent to a wedge of spheres. The number and dimension of the spheres in the wedge are determined, and (partially conjectural) links to other combinatorially defined complexes are described. In addition we study for positive integers r,s and k the simplicial complex of all bipartite graphs G on bipartition such that there is no matching of size k in G, and obtain results similar to those obtained for . S. Linusson and V. Welker supported by EC’s IHRP program through grant HPRN-CT-2001-00272. J. Shareshian partially supported by National Science Foundation grants DMS-0070757 and DMS-0030483.     

A new device to study ex-vivo the effects of extracorporeal photochemotherapy on the immune system

Extracorporeal photochemotherapy (ECP) is a medical procedure effective in the treatment of several different T-cell mediated diseases such as cutaneous T-cell lymphoma and Graft-versus-Host Disease. During ECP treatment the patient's blood is processed by means of a cell separator to collect leukocytes (leukapheresis), mostly lymphocytes and monocytes, which are then incubated with the photoactive drug 8-methoxypsoralen (8-MOP), exposed to ultraviolet-A light (UV-A) and reinfused to the patient. It has been suggested that during ECP not only UV-A irradiation but also changes in the environmental condition may be relevant. Although ECP has been shown to have an in-vivo immunomodulatory effect, the mechanisms through which ECP exerts its effect remain elusive. One of the reasons for this incomplete knowledge is the absence of a reliable model for ECP. In order to investigate the effect of ECP on the peripheral immune system, we developed a new device which mimics the complete ECP cycle including blood transit through the cell separator. Peripheral blood samples (50ml) were obtained from volunteers and processed using a peristaltic pump. Peripheral blood mononuclear cells (PBMC) were then collected and treated with 8-MOP and UV-A under the same conditions used for the patients' therapy. Using this strategy we investigated 8-MOP, UV-A and their combined effect on the production of the pro-inflammatory cytokines interferon-gamma (IFN-gamma), interleukine-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) in PBMC with and without polyclonal stimulation. We firstly demonstrated that our device does not affect total red and white blood cell counts. After 8-MOP and UV-A irradiation a significant decrease was observed in both activated CD4(+) and CD8(+) T lymphocytes producing IFN-gamma, IL-2 and TNF-alpha. Our findings are in line with those previously obtained in humans after complete ECP treatment, thus suggesting that our newly developed device is suitable for investigating the mechanism of action of ECP ex-vivo.