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In the past decade, tissue engineering has evolved from a promising technology to an established scientific field. Large attention has focussed on developing scaffolds from both biodegradable and nondegradable polymers to be cultivated with cells, to replace human body defects. The major drawback of most polymers is however their limited cell-interactive properties. An additional complication when developing a surface modification protocol for those materials is the transferability of protocols from 2D substrates to 3D scaffolds. In the present work, we therefore report on possible biological effects originating from the transfer of a double protein coating protocol, involving gelatin type B and fibronectin, from 2D poly-ε-caprolactone (PCL) films to 3D PCL scaffolds produced by rapid prototyping. A variety of techniques including scanning electron microscopy, X-ray photoelectron spectroscopy and confocal fluorescence microscopy confirmed a successful and homogeneous protein-coating on both 2D and 3D substrates. Interestingly, the biological performance of the double protein-coated PCL substrates, reflected by the initial cell adhesion, proliferation, and colonization was superior compared to the other surface modification steps, independent of the material dimension. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012. [ABSTRACT FROM AUTHOR]

The probability of skipping a word is influenced by its processing ease. For instance, a word that is predictable from the preceding context is skipped more often than an unpredictable word. A meta-analysis of studies examining this predictability effect reported effect sizes ranging from 0 to 13%, with an average of 8%. One study does not fit within this picture and reported 23% more skipping of Dutch pronouns in sentences in which the pronoun had no disambiguating value (e.g. 'Mary was envious of Helen because she never looked so good') than in sentences where it did have a disambiguating value (e.g. 'Mary was envious of Albert because she never looked so good'). We re-examined this ambiguity in Dutch using a task that more closely resembles normal reading and observed only a 9% difference in skipping of the pronoun, bringing this linguistic effect in line with the other findings. [ABSTRACT FROM AUTHOR]

Abstract: An in silico survey of all known 3D-structures of glycoside hydrolases that contain a ligand in the −1 subsite is presented. A recurrent crucial positioning of active site residues indicates a common general strategy for electrostatic stabilisation directed to the carbohydrate’s ring-oxygen at the transition state. This is substantially different depending on whether the enzyme’s proton donor is syn or anti positioned versus the substrate. A comprehensive list of enzymes belonging to 42 different families is given and selected examples are described. An implication for an early evolution scenario of glycoside hydrolases is discussed. [Copyright &y& Elsevier]

An in silico survey of the −1 subsite of all known 3D-structures of O-glycoside hydrolases containing a suitably positioned ligand has led to the recognition – apparently without exceptions – of a transition state stabilising hydrophobic platform which is complementary to a crucial hydrophobic patch of the ligand. This platform is family-specific and highly conserved. A comprehensive list is given with examples of enzymes belonging to 33 different families. Several typical constellations of platform – protein residues are described. [Copyright &y& Elsevier]

Trehalose (α-D-glucopyranosyl-(1,1)-α-D-glucopyranoside) is widely used in the food industry, thanks to its protective effect against freezing and dehydration. Analogs of trehalose have the additional benefit that they are not digested and thus do not contribute to our caloric intake. Such trehalose analogs can be produced with the enzyme trehalose phosphorylase, when it is applied in the reverse, synthetic mode. Despite the enzyme's broad acceptor specificity, its catalytic efficiency for alternative monosaccharides is much lower than for glucose. For galactose, this difference is shown here to be caused by a lower K(m) whereas the k(cat) for both substrates is equal. Consequently, increasing the affinity was attempted by enzyme engineering of the trehalose phosphorylase from Thermoanaerobacter brockii, using both semirational and random mutagenesis. While a semirational approach proved unsuccessful, high-throughput screening of an error-prone PCR library resulted in the discovery of three beneficial mutations that lowered K(m) two- to three-fold. In addition, it was found that mutation of these positions also leads to an improved catalytic efficiency for mannose and fructose, suggesting their involvement in acceptor promiscuity. Combining the beneficial mutations did not further improve the affinity, and even resulted in a decreased catalytic activity and thermostability. Therefore, enzyme variant R448S is proposed as new biocatalyst for the industrial production of lactotrehalose (α-D-glucopyranosyl-(1,1)-α-D-galactopyranoside)., (Copyright © 2012 American Institute of Chemical Engineers (AIChE).)

Cellodextrin phosphorylase from Clostridium stercorarium has been recombinantly expressed in Escherichia coli for the first time. Kinetic characterization of the purified enzyme has revealed that aryl and alkyl β-glucosides can be efficiently glycosylated, an activity that has not yet been described for this enzyme class. To obtain a better understanding of the factors that determine the enzyme's specificity, homology modeling and ligand docking were applied. Residue W168 has been found to form a hydrophobic stacking interaction with the substrate in subsite +2, and its importance has been examined by means of site-directed mutagenesis. The mutant W168A retains about half of its catalytic activity, indicating that other residues also contribute to the binding affinity of subsite +2. Finally, residue D474 has been identified as the catalytic acid, interacting with the glycosidic oxygen between subsites -1 and +1. Mutating this residue results in complete loss of activity. These results, for the first time, provide an insight in the enzyme-substrate interactions that determine the activity and specificity of cellodextrin phosphorylases., (Copyright © 2011 American Institute of Chemical Engineers (AIChE).)

Disaccharide phosphorylases are able to catalyze both the synthesis and the breakdown of disaccharides and have thus emerged as attractive platforms for tailor-made sugar synthesis. Cellobiose phosphorylase from Cellulomonas uda (CPCuda) is an enzyme that belongs to glycoside hydrolase family 94 and catalyzes the reversible breakdown of cellobiose [beta-D-glucopyranosyl-(1,4)-D-glucopyranose] to alpha-D-glucose-1-phosphate and D-glucose. Crystals of ligand-free recombinant CPCuda and of its complexes with substrates and reaction products yielded complete X-ray diffraction data sets to high resolution using synchrotron radiation but suffered from significant variability in diffraction quality. In at least one case an intriguing space-group transition from a primitive monoclinic to a primitive orthorhombic lattice was observed during data collection. The structure of CPCuda was determined by maximum-likelihood molecular replacement, thus establishing a starting point for an investigation of the structural and mechanistic determinants of disaccharide phosphorylase activity.

Class I alpha-mannosidases (glycoside hydrolase family GH47) play key roles in the maturation of N-glycans and the ER-associated degradation of unfolded glycoproteins. The 1.95 A resolution structure of a fungal alpha-1,2-mannosidase in complex with the substrate analogue methyl-alpha-D-lyxopyranosyl-(1',2)-alpha-D-mannopyranoside (LM) shows the intact disaccharide spanning the -1/+1 subsites, with the D-lyxoside ring in the -1 subsite in the 1C4 chair conformation, and provides insight into the mechanism of catalysis. The absence of the C5' hydroxymethyl group on the D-lyxoside moiety results in the side chain of Arg407 adopting two alternative conformations: the minor one interacting with Asp375 and the major one interacting with both the D-lyxoside and the catalytic base Glu409, thus disrupting its function. Chemical modification of Asp375 has previously been shown to inactivate the enzyme. Taken together, the data suggest that Arg407, which belongs to the conserved sequence motif RPExxE, may act to modulate the activity of the enzyme. The proposed mechanism for modulating the activity is potentially a general mechanism for this superfamily.