Novel findings on the metabolic effects of the low glycaemic carbohydrate isomaltulose (Palatinose™)

The slow digestible disaccharide isomaltulose (iso; Palatinose™) is available as novel functional carbohydrate ingredient for manufacturing of low glycaemic foods and beverages. Although basically characterised, various information on physiological effects of iso are still lacking. Thus, the objective of the present study was to expand scientific knowledge of physiological characteristics of iso by a set of three human intervention trials. Using an ileostomy model, iso was found to be essentially absorbed, irrespective of the nature of food (beverage and solid food). Apparent digestibility of 50 g iso from two different meals was 95·5 and 98·8 %; apparent absorption was 93·6 and 96·1 %, respectively. In healthy volunteers, a single dose intake of iso resulted in lower postprandial blood glucose and insulin responses than did sucrose (suc), while showing prolonged blood glucose delivery over 3 h test. In a 4-week trial with hyperlipidaemic individuals, regular consumption of 50 g/d iso within a Western-type diet was well tolerated and did not affect blood lipids. Fasting blood glucose and insulin resistance were lower after the 4-week iso intervention compared with baseline. This would be consistent with possible beneficial metabolic effects as a consequence of the lower and prolonged glycaemic response and lower insulinaemic burden. However, there was no significant difference at 4 weeks after iso compared with suc. In conclusion, the study shows that iso is completely available from the small intestine, irrespective of food matrix, leading to a prolonged delivery of blood glucose. Regular iso consumption is well tolerated also in subjects with increased risk for vascular diseases

Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve – especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4high/CD25high/CD45RAhigh ‘regulatory T cells’ and CD8high/CD62Lhigh/CD45RAneg ‘central memory T cells’, have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research

Fermenter Production of an Artificial Fab Fragment, Rationally Designed for the Antigen Cystatin, and Its Optimized Crystallization Through Constant Domain Shuffling

The synthetic antibody model "M41" was rationally designed with a binding site complementary to chicken egg white cystatin as the prescribed antigen. In order to permit comparison between the computer model and an experimental three-dimensional structure of the artificial protein, its X-ray crystallographic analysis was pursued. For this purpose, M41 was expressed as a recombinant Fab fragment in E. coli by medium cell density fermentation employing the tightly regulated tetracycline promoter. The Fab fragment was efficiently purified via a His-6 tail fused to its heavy chain and immobilized metal affinity chromatography. To raise the chances for the productive formation of crystal packing contacts, three versions of the Fab fragment were generated with differing constant domains. One of these, the variant with murine CK and CH1γ1 domains, was successfully crystallized by microseeding in a sitting drop. The orthorhombic crystals exhibited symmetry of the space group P212121 with unit cell dimensions a = 104.7 A, b = 113.9 A, c = 98.8 A and diffracted X-rays to a nominal resolution of 2.5 A

The rational construction of an antibody against cystatin: lessons from the crystal structure of an artificial F_ab Fragment

In a protein design study the artificial antibody M41 was modelled with its binding site complementary to the protease inhibitor cystatin, which was chosen as a structurally well-characterized “antigen”. The modelling of M41 took advantage of the crystal structure of the anti-lysozyme antibody HyHEL-10 as a structural template. Its combining site was reshaped by replacing 19 amino acid side-chains in the hypervariable loops. In addition, ten amino acid residues were substituted in the framework regions. The crystal structure of the corresponding antibody model M41, which was produced as an Fab fragment in Escherichia coli, was determined at a resolution of 1.95 Å. The crystals exhibited symmetry of the space group P212121 (a = 96.5 Å; b = 103.5 Å; c = 113.6 Å) with two Fab fragments in the asymmetric unit, which were independently refined (final R-factor 21.7%). The resulting coordinates were used for a detailed comparison with the modelled protein structure. It was found that the mutual arrangement of the six complementarity-determining regions as well as most of their backbone conformation had been correctly predicted. One major difference that was detected for the conformation of a five residue insertion in complementarity-determining region L1 could be explained by an erroneously defined segment in the structure of the antibody 4-4-20, which had been used as a template for this loop. In the light of more recent crystallographic data it appears that this segment adopts a new canonical structure. Apart from this region, most of the side-chains in the antigen-binding site had been properly placed in the M41 model. There was however one important exception concerning Trp H98, whose side-chain conformation had been kept as it appeared in HyHEL-10. The differing orientation of this residue in the model compared with the crystal structure of the artificial Fab fragment M41 explains why an antigen affinity could not be detected so far. The detailed analysis of this and other, more subtle deviations suggests how to make this Fab fragment function by introducing a few additional amino acid changes into M41