Joost L. J. van Dongen

Protein dimerization induced by supramolecular interactions with cucurbit[8]uril

Hosted dimerization: Proteins such as yellow fluorescent protein (YFP) with an N-terminal FGG peptide motif form dimers mediated by supramolecular interactions with cucurbit[8]uril (see scheme). The protein dimerization, which is observed by FRET and size-exclusion chromatography, can be reversed with methyl viologen as a bioorthogonal ligand, which displaces the FGG motifs from the cucurbit[8]uril host. Ein Platzchen zum Kuppeln: Proteine wie das gelb fluoreszierende Protein (YFP), die ein N-terminales FGG-Motiv tragen, dimerisieren unter Mitwirkung von supramolekularen Wechselwirkungen innerhalb des Cucurbit[8]uril-Rings (siehe Schema). Die durch FRET und Grosenausschlusschromatographie beobachtbare Dimerisierung lasst sich durch Zugabe von Methylviologen umkehren, das als bioorthogonaler Ligand die FGG-Motive aus dem Cucurbit[8]uril-Wirt verdrangt.

Macrocyclization of enzyme-based supramolecular polymers

AB type monomers for supramolecular polymers have been developed based on the strong and reversible noncovalent interaction between ribonuclease S-peptide (A) and S-protein (B), resulting in an active enzyme complex as the linking unit. Two AB-type protein constructs are synthesized differing in the length of the flexible oligo(ethylene glycol) spacer separating the two end groups. Using an experimental setup where size exclusion chromatography is directly coupled to Q-TOF mass spectrometry, we have analyzed the self-assembled architectures as a function of concentration. The theory of macrocyclization under thermodynamic control is used to quantitatively analyze the experimental data. Using this theory, we show that AB-type monomers linked by flexible linkers grow reversibly via ring–chain competition. Inherently the formation of linear polymeric assemblies is beyond the capability of these types of building blocks due to concentration limits of proteins. The results therefore contribute to the general understanding of supramolecular polymerization with biological building blocks and demonstrate design requirements for monomers if linear polymerization is desired.

Exercise-induced oxidative stress in older adults as a function of habitual activity level.

OBJECTIVES It has been suggested that regular physical activity might maintain and promote the antioxidant defense capacity against oxidative stress. Therefore, we assessed exercise-induced oxidative stress in relation to habitual physical activity level (PAL) in older adults. DESIGN The study included a 2-week observation period for the measurement of average daily metabolic rate (ADMR) and PAL. Exercise-induced oxidative stress was measured during a 45-minute cycling test at submaximal intensity. SETTING A university medical research center. PARTICIPANTS Twenty-six subjects volunteered for the study (n = 26; mean age +/- standard deviation 60 +/- 1; body mass index 27 +/- 1 kg/m2). MEASUREMENTS PAL was determined as ADMR combined with a measurement of basal metabolic rate (BMR): PAL = ADMR/BMR. ADMR was measured over 2 weeks with the doubly labeled water method, preceded by a BMR measurement with a ventilated hood. Antipyrine oxidation was used as marker for oxidative stress in vivo. Reaction of antipyrine with hydroxyl radicals results in the formation of para-hydroxyantipyrine (p-APOH) and ortho-hydroxyantipyrine (o-APOH), where o-APOH is not formed through alternative oxygenetic pathways. RESULTS PAL was inversely related to the exercise-induced increase in the ratio of o-APOH to native antipyrine (r = 0.49, P = .010). The relationship between PAL and exercise-induced increase in the ratio of p-APOH (r = 0.30, P = .140) or thiobarbituric acid reactive species (r = 0.31, P = .130) did not reach the level of significance. CONCLUSION Physically active older adults have a reduced exercise-induced oxidative stress than older adults with a lower level of physical activity. It seems that regular physical activity improves the antioxidant defense capacity.

Off-line size-exclusion chromatographic fractionation-matrix-assisted laser desorption ionization-time-of-flight mass spectrometry for polymer characterization. Theoretical and experimental study

The parameters affecting the fractionation performance in size-exclusion chromatography (SEC) of broad polymer samples were investigated. Some equations were derived which enable the prediction of polydispersity (PD) in an SEC fraction. Good agreements were obtained between the calculated data and the experimental values. Based on these equations, SEC fractionation conditions were optimized. In the off-line SEC-matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), two different modes can be employed, i.e., using MALDI-MS to provide an absolute calibration curve for SEC, or using SEC as a sample preparation step for MALDI-MS measurements. It was demonstrated that it is more reliable to use the latter combination, because most problems inherent in SEC can be circumvented. Some guidelines for the optimization of off-line SEC fractionation-MALDI-TOF-MS were given. It was found that under optimized conditions normally only a few SEC fractions are already sufficient to separate a highly polydisperse sample into portions of low PD that can accurately be measured by MALDI-TOF-MS.

Generation of CsI cluster ions for mass calibration in matrix-assisted laser desorption/ionization mass spectrometry

A simple method was developed for the generation of cesium iodide (CsI) cluster ions up to m/z over 20,000 in matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Calibration ions in both positive and negative ion modes can readily be generated from a single MALDI spot of CsI3 with 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene] malononitrile (DCTB) matrix. The major cluster ion series observed in the positive ion mode is [(CsI)nCs]+, and in the negative ion mode is [(CsI)nI]−. In both cluster series, ions spread evenly every 259.81 units. The easy method described here for the production of CsI cluster ions should be useful for MALDI MS calibrations.

Non-aqueous capillary electrophoresis using non-dissociating solvents. Application to the separation of highly hydrophobic oligomers

The application of non-aqueous capillary electrophoresis for the separation of very hydrophobic oligomers has been studied. N-Phenylaniline oligomers having degrees of polymerisation (n) of 2, 4, 6, and 8 were taken as model compounds. Capillary electrophoresis could be performed using a mixture of non-aqueous solvents with a high percentage of solvents with a low dielectric constant. These solvents, such as tetrahydrofuran (THF), chloroform or dichloromethane, are needed to solubilise the hydrophobic solutes in the electrolyte. The composition of the solvent mixture and the nature of the acid added to the electrolyte, which is needed to obtain electrophoretic motion of the N-phenylaniline oligomers, are discussed in detail. Next, other parameters such as ionic strength, injection time, electric field, and temperature were investigated too and their influence on the separation is discussed as well. The existence of a reversed (anodic) electroosmotic flow in a fused-silica capillary containing a THF-methanol mixture under acidic conditions is reported.

Noncovalent Synthesis of Protein Dendrimers

The covalent synthesis of complex biomolecular systems such as multivalent protein dendrimers often proceeds with low efficiency, thereby making alternative strategies based on noncovalent chemistry of high interest. Here, the synthesis of protein dendrimers using a strong but noncovalent interaction between a peptide and complementary protein is proposed as an efficient strategy to arrive at dendrimers fully functionalized with protein domains. The association of S-peptide to S-protein results in the formation of an active enzyme (ribonuclease S) and therefore serves as an ideal system to explore this synthetic approach. Native chemical ligation was used to couple four S-peptides by means of their C-terminal thioester to a cysteine-functionalized dendritic scaffold, thus yielding a tetravalent S-peptide wedge. A fully functional ribonuclease S tetramer was prepared by addition of four equivalents of S-protein. Biophysical techniques (isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and mass spectrometry) and an enzymatic activity assay were used to verify the formation of the multivalent complex. The noncovalent synthetic strategy presented here provides access to well-defined, dynamic, semisynthetic protein assemblies in high yield and is therefore of interest to the field of nanomedicine as well as biomaterials.

Metallo-supramolecular oligo(p-phenylene vinylene)/[60]fullerene architectures: towards functional materials

The assembly of p-conjugated woligo(p-phenylene vinylene)x OPV donors and w60xfullerene acceptor moieties into a supramolecular donor-acceptor system is achieved by ruthenium complexation. The synthesis and optical properties of a novel photoactive supramolecular dyad and triad are described to access the photoinduced formation of a charge-separated state. 2002 Elsevier Science B.V. All rights reserved.

Photodimerization Processes in Self‐Assembled Chiral Oligo(p‐phenylenevinylene) Bolaamphiphiles

An oligo(p-phenylene vinylene) (OPV) amphiphile has been synthesized with a positively charged head group on one end and a hydrophilic ethyleneglycol wedge on the other. In water, this bolaamphiphile forms vesicles consisting of a monolayer in which the OPV surfactants are organized in a helical head-to-tail fashion. Mass spectrometry analysis and reversed-phase high performance liquid chromatography (RP-HPLC) reveals that because of the presence of two double bonds in the OPV units, photo-induced [2+2] cycloaddition takes place resulting in polymerization of the surfactants. Performing RP-HPLC allowed the OPV dimer to be isolated in a sufficient amount to perform a variety of one- and two-dimensional NMR experiments. These experiments show a highly stereoselective photochemical cycloaddition process resulting in a regioselective head-to-tail dimerization with a stereoselective syn arrangement.

Therapeutic drug monitoring of infliximab: performance evaluation of three commercial ELISA kits

Abstract Background: Therapeutic drug monitoring (TDM) of infliximab (IFX, Remicade®) can aid to optimize therapy efficacy. Many assays are available for this purpose. However, a reference standard is lacking. Therefore, we evaluated the analytical performance, agreement and clinically relevant differences of three commercially available IFX ELISA kits on an automated processing system. Methods: The kits of Theradiag (Lisa Tracker Infliximab), Progenika (Promonitor IFX) and apDia (Infliximab ELISA) were implemented on an automated processing system. Imprecision was determined by triplicate measurements of patient samples on five days. Agreement was evaluated by analysis of 30 patient samples and four spiked samples by the selected ELISA kits and the in-house IFX ELISA of Sanquin Diagnostics (Amsterdam, The Netherlands). Therapeutic consequences were evaluated by dividing patients into four treatment groups using cut-off levels of 1, 3 and 7 μg/mL and determining assay concordance. Results: Within-run and between-run imprecision were acceptable (≤12% and ≤17%, respectively) within the quantification range of the selected ELISA kits. The apDia assay had the best precision and agreement to target values. Statistically significant differences were found between all assays except between Sanquin Diagnostics and the Lisa Tracker assay. The Promonitor assay measured the lowest IFX concentrations, the apDia assay the highest. When patients were classified in four treatment categories, 70% concordance was achieved. Conclusions: Although all assays are suitable for TDM, significant differences were observed in both imprecision and agreement. Therapeutic consequences were acceptable when patients were divided in treatment categories, but this could be improved by assay standardization.