Accelerated physical ageing of poly(1,4-cyclohexylenedimethylene-co-2,2,4,4-tetramethyl-1,3-cyclobutanediol terephthalate).

Successfully evaluating plastic lifetime requires understanding of the relationships between polymer dynamics and mechanical performance as a function of thermal ageing. The relatively high T g (T g = 110 °C) of poly(1,4-cyclohexylenedimethylene-co-2,2,4,4-tetramethyl-1,3-cyclobutanediol terephthalate) (PCTT) renders it useful as a substituent for PET in higher temperature applications. This work links thermal ageing and mechanical performance of a commercial PCTT plastic after exposure to 40-80 °C for up to 2950 h. No chemical or conformational changes were found while pronounced physical ageing, measured as enthalpic relaxation, caused yield hardening (28% increase in yield strength) and embrittlement (80% decrease in toughness). Enthalpic relaxation increased with temperature and time to 3.8 J g-1 and correlated to the determined toughness and yield strength. Finally, a 9% increase in Young's modulus was observed independent of temperature and with no correlation to enthalpic relaxation. Enthalpic relaxation followed Vogel-Fulcher-Tammann behaviour, while yield strength and charpy v-notch toughness followed Arrhenius behaviour enabling prediction of the different properties with time and temperature.

Crystal structure of quinone-dependent alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes. A versatile dehydrogenase oxidizing alcohols and carbohydrates.

The quinone-dependent alcohol dehydrogenase (PQQ-ADH, E.C. 1.1.5.2) from the Gram-negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three-dimensional (3D) structures of the native form, with PQQ and a Ca(2+) ion, and of the enzyme in complex with a Zn(2+) ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ-ADH displays an eight-bladed ß-propeller fold, characteristic of Type I quinone-dependent methanol dehydrogenases. However, three of the four ligands of the Ca(2+) ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ-ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ-dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer.

A novel type carbohydrate-binding module identified in alpha-glucan, water dikinases is specific for regulated plastidial starch metabolism.

The phosphorylation of the amylopectin fraction of starch catalyzed by the alpha-glucan, water dikinase (GWD, EC 2.7.9.4) plays a pivotal role in starch metabolism. Limited proteolysis of the potato tuber (Solanum tuberosum) GWD (StGWD, 155 kDa) by trypsin primarily produced stable fragments of 33 and 122 kDa, termed the SBD fragment and N11, respectively, as generated by trypsin cleavage at Arg-286. SBD and N11 were generated using recombinant DNA technology and purified to near homogeneity. Tandem repeat sequences, SBD-1 and SBD-2, of a region that is significantly similar in sequence to N-terminal regions of plastidial alpha-amylases are located in the N-terminus of StGWD. The SBD-1 motif is located within the sequence of the SBD fragment, and our results demonstrate that the fragment composes a new and novel carbohydrate-binding module (CBM), apparently specific for plastidial alpha-glucan degradation. By mutational analyses of conserved Trp residues located within the SBD-1 motif, W62 and W117, we show that these aromatic residues are vital for carbohydrate binding. N11 still possessed starch phosphorylating activity, but with a 2-fold higher specific activity compared to that of wild type (WT) StGWD using potato starch as the glucan substrate, whereas it had double the K(m) value for the same substrate. Furthermore, investigation of the chains phosphorylated by WT StGWD and N11 shows that N11 exhibits a higher preference for phosphorylating shorter chains of the amylopectin molecule as compared to WT. From analyses of the glucan substrate specificity, we found up to 5-fold higher specific activity for N11 using amylose as the substrate.

Small-number statistics near the clustering transition in a compartmentalized granular gas.

Statistical fluctuations are observed to profoundly influence the clustering behavior of granular material in a vibrated system consisting of two connected compartments. When the number of particles N is sufficiently large ( N approximately 300 is sufficient), the clustering follows the lines of a standard second-order phase transition and a mean-field description works. For smaller N , however, the enhanced influence of statistical fluctuations breaks the mean-field behavior. We quantitatively describe the competition between fluctuations and mean-field behavior (as a function of N ) using a dynamical flux model and molecular dynamics simulations.

A novel isoform of glucan, water dikinase phosphorylates pre-phosphorylated alpha-glucans and is involved in starch degradation in Arabidopsis.

An Arabidopsis thaliana gene encoding a homologue of the potato alpha-glucan, water dikinase GWD, previously known as R1, was identified by screening the Arabidopsis genome and named AtGWD3. The AtGWD3 cDNA was isolated, heterologously expressed and the protein was purified to apparent homogeneity to determine the enzymatic function. In contrast to the potato GWD protein, the AtGWD3 primarily catalysed phosphorylation at the C-3 position of the glucose unit of preferably pre-phosphorylated amylopectin substrate with long side chains. An Arabidopsis mutant, termed Atgwd3, with downregulated expression of the AtGWD3 gene was analysed. In Atgwd3 the amount of leaf starch was constantly higher than wild type during the diurnal cycle. Compared with wild-type leaf starch, the level of C-3 phosphorylation of the glucosyl moiety of starch in this mutant was reduced. Taken together, these data indicate that the C-3 linked phospho-ester in starch plays a so far unnoticed specific role in the degradation of transitory starch.

Alpha-glucan, water dikinase (GWD): a plastidic enzyme with redox-regulated and coordinated catalytic activity and binding affinity.

The recently discovered potato tuber (Solanum tuberosum) alpha-glucan, water dikinase (GWD) (formerly known as R1) catalyzes the phosphorylation of starch by a dikinase-type reaction mechanism in which the beta-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glucosyl residue of starch. In the present study, we found that the GWD enzyme is inactive in the oxidized form, which is accompanied by the formation of a specific intramolecular disulfide bond as determined by disulfide-linked peptide mapping. The regulatory properties of this disulfide linkage were confirmed by site-directed mutagenesis studies. Both reduced thioredoxin (Trx) f and Trx m from spinach leaves reduced and activated oxidized GWD at very low concentrations, with Trx f being the more efficient, yielding an S0.5 value of 0.4 microM. Interestingly, GWD displays a reversible and selective binding to starch granules depending on the illumination state of the plant. Here we show that starch granule-bound GWD isolated from dark-adapted plants exists in the inactive, oxidized form, which is capable of reactivation upon treatment with reduced Trx. Furthermore, the soluble form of GWD was found in its fully reduced state, providing evidence of a Trx-controlled regulation mechanism linking enzymatic activity and specific binding affinities of a protein to an intracellular surface. The regulatory site sequence, CFATC, of potato GWD is conserved in chloroplast-targeted GWDs from other species, suggesting an overall redox regulation of the GWD enzyme.

Functional domain organization of the potato alpha-glucan, water dikinase (GWD): evidence for separate site catalysis as revealed by limited proteolysis and deletion mutants.

The potato tuber (Solanum tuberosum) GWD (alpha-glucan, water dikinase) catalyses the phosphorylation of starch by a dikinase-type reaction mechanism in which the beta-phosphate of ATP is transferred to the glucosyl residue of amylopectin. GWD shows sequence similarity to bacterial pyruvate, water dikinase and PPDK (pyruvate, phosphate dikinase). In the present study, we examine the structure-function relationship of GWD. Analysis of proteolytic fragments of GWD, in conjunction with peptide microsequencing and the generation of deletion mutants, indicates that GWD is comprised of five discrete domains of 37, 24, 21, 36 and 38 kDa. The catalytic histidine, which mediates the phosphoryl group transfer from ATP to starch, is located on the 36 kDa fragment, whereas the 38 kDa C-terminal fragment contains the ATP-binding site. Binding of the glucan molecule appears to be confined to regions containing the three N-terminal domains. Deletion mutants were generated to investigate the functional interdependency of the putative ATP- and glucan-binding domains. A truncated form of GWD expressing the 36 and 38 kDa C-terminal domains was found to catalyse the E+ATP-->E-P+AMP+P(i) (where P(i) stands for orthophosphate) partial reaction, but not the E-P+glucan-->E+glucan-P partial reaction. CD experiments provided evidence for large structural changes on autophosphorylation of GWD, indicating that GWD employs a swivelling-domain mechanism for enzymic phosphotransfer similar to that seen for PPDK.

Impact on soft sand: void collapse and jet formation.

Very fine sand is prepared in a well-defined and fully decompactified state by letting gas bubble through it. After turning off the gas stream, a steel ball is dropped on the sand. On impact of the ball, sand is blown away in all directions ("splash") and an impact crater forms. When this cavity collapses, a granular jet emerges and is driven straight into the air. A second jet goes downwards into the air bubble entrained during the process, thus pushing surface material deep into the ground. The air bubble rises slowly towards the surface, causing a granular eruption. In addition to the experiments and the discrete particle simulations we present a simple continuum theory to account for the void collapse leading to the formation of the upward and downward jets.

Competitive clustering in a bidisperse granular gas: experiment, molecular dynamics, and flux model.

A compartmentalized bidisperse granular gas clusters competitively [Phys. Rev. Lett. 89, 214301 (2002)]]: By tuning the shaking strength, the clustering can be directed either towards the compartment initially containing mainly small particles or to the compartment containing mainly large particles. Here, the conditions under which this competitive clustering occurs are studied experimentally, numerically (by means of molecular dynamics simulations), and analytically. A minimal model is derived that quantitatively accounts for the observed phenomena.

Functional characterization of alpha-glucan,water dikinase, the starch phosphorylating enzyme.

GWD (alpha-glucan,water dikinase) is the enzyme that catalyses the phosphorylation of starch by a dikinase-type reaction in which the beta-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glycosyl residue of amylopectin. GWD shows similarity in both sequence and reaction mechanism to bacterial PPS (pyruvate,water dikinase) and PPDK (pyruvate,phosphate dikinase). Amino acid sequence alignments identified a conserved histidine residue located in the putative phosphohistidine domain of potato GWD. Site-directed mutagenesis of this histidine residue resulted in an inactive enzyme and loss of autophosphorylation. Native GWD is a homodimer and shows a strict requirement for the presence of alpha-1,6 branch points in its polyglucan substrate, and exhibits a sharp 20-fold increase in activity when the degree of polymerization is increased from 27.8 to 29.5. In spite of the high variability in the degree of starch phosphorylation, GWD proteins are ubiquitous in plants. The overall reaction mechanism of GWD is similar to that of PPS and PPDK, but the GWD family appears to have arisen after divergence of the plant kingdom. The nucleotide-binding domain of GWD exhibits a closer phylogenetic relationship to prokaryotic PPSs than to PPDKs.

Influence of solitons on the transition to spatiotemporal chaos in coupled map lattices.

We study the transition from laminar to chaotic behavior in deterministic chaotic coupled map lattices and in an extension of the stochastic Domany-Kinzel cellular automaton [E. Domany and W. Kinzel, Phys. Rev. Lett. 53, 311 (1984)]. For the deterministic coupled map lattices, we find evidence that "solitons" can change the nature of the transition: for short soliton lifetimes it is of second order, while for longer but finite lifetimes, it is more reminiscent of a first-order transition. In the second-order regime, the deterministic model behaves like directed percolation with infinitely many absorbing states; we present evidence obtained from the study of bulk properties and the spreading of chaotic seeds in a laminar background. To study the influence of the solitons more specifically, we introduce a soliton including variant of the stochastic Domany-Kinzel cellular automaton. Similar to the deterministic model, we find a transition from second- to first-order behavior due to the solitons, both in a mean-field analysis and in a numerical study of the statistical properties of this stochastic model. Our study illustrates that under the appropriate mapping some deterministic chaotic systems behave like stochastic models; but it is hard to know precisely which degrees of freedom need to be included in such description.

Competitive clustering in a bidisperse granular gas.

A bidisperse granular gas in a compartmentalized system is experimentally found to cluster competitively: Depending on the shaking strength, the clustering can be directed either towards the compartment initially containing mainly small particles or to the one containing mainly large particles. The experimental observations are quantitatively explained within a flux model.

"Sausage-string" appearance of arteries and arterioles can be caused by an instability of the blood vessel wall.

Vascular damage induced by acute hypertension is preceded by a peculiar pattern where blood vessels show alternating regions of constrictions and dilations ("sausages on a string"). The pattern occurs in the smaller blood vessels, and it plays a central role in causing the vascular damage. A related vascular pattern has been observed in larger vessels from several organs during angiography. In the larger vessels the occurrence of the pattern does not appear to be related to acute hypertension. A unifying feature between the phenomenon in large and small vessels seems to be an increase in vascular wall tension. Despite much research, the mechanisms underlying the sausage pattern have remained unknown. Here we present an anisotropic model of the vessel wall and show that the sausage pattern can arise because of an instability of the vessel wall. The model reproduces many of the key features observed experimentally. Most importantly, it suggests that the "sausaging" phenomenon is neither caused by a mechanical failure of the vessel wall due to a high blood pressure nor is it due to standing pressure waves caused by the beating of the heart. Rather, it is the expression of a general instability phenomenon. Experimental data suggest that the structural changes induced by the instability may cause secondary damage to the wall of small arteries and arterioles in the form of endothelial hyperpermeability followed by local fibrinoid necrosis of the vascular wall.

Starch phosphorylation: a new front line in starch research.

Starch is the primary energy reserve in higher plants and is, after cellulose, the second most abundant carbohydrate in the biosphere. It is also the most important energy source in the human diet and, being a biodegradable polymer with well-defined chemical properties, has an enormous potential as a versatile renewable resource. The only naturally occurring covalent modification of starch is phosphorylation. Starch phosphate esters were discovered a century ago but were long regarded as a curiosity, receiving little attention. Indeed, the mechanism for starch phosphorylation remained completely unknown until recently. The starch-phosphorylating enzyme is an alpha-glucan water dikinase. It is now known that starch phosphorylation plays a central role in starch metabolism.

Truncation of the amino terminus of branching enzyme changes its chain transfer pattern.

Previous work has reported the production of an Escherichia coli branching enzyme with a 112-residue deletion at the amino terminal by limited proteolysis. Here, we study the chain transfer pattern of this enzyme. Gel-permeation chromatography of in vitro branched amylose shows that the truncated branching enzyme transfers fewer short chains (degree of polymerization [d.p.] <20) and a greater proportion of intermediate size chains (d.p. 30-90) than the native enzyme. High-performance anion-exchange chromatography (HPAEC) of the branching limited alpha-glucan product indicates that the truncated branching enzyme transfers a smaller proportion of chains with d.p. 4-11 and more chains longer than d.p. 12. Also, the genes encoding native or truncated branching enzyme were individually expressed in a branching enzyme-deficient mutant, AC71 (glgB(-)). By HPAEC analysis of the purified alpha-glucans we find that truncated branching enzyme transfers fewer chains of d.p. 5-11 and more chains longer than d.p. 12 relative to the full-length enzyme. These observations allow us to conclude that truncation of the amino-terminal domain has altered the branching pattern of the enzyme. Our results are consistent with the construction of hybrid branching enzymes from the maize isoforms.