Canards of mixed type in a neural burster.

Canards are solutions of slow-fast systems that spend long times near branches of repelling equilibria, periodic orbits, or higher-dimensional invariant sets. Here, we report on the observation of a new type of canard orbit, labeled a canard of mixed type. This canard orbit is a hybrid of the classical limit cycle canards, which spend long times near attracting and repelling branches of equilibria, and torus canards, which spend long times near attracting and repelling branches of periodic orbits. The canards of mixed type arise in a model of neural bursting activity of fold-fold cycle type, and, as other canard phenomena, separate different dynamic states.

Exploring and exploiting starch-modifying amylomaltases from thermophiles.

Starch is a staple food present in water-insoluble granules in many economically important crops. It is composed of two glucose polymers: the linear alpha-1,4-linked amylose and amylopectin with a backbone of alpha-1,4-glycosidic bonds and alpha-1,6-linked side chains. To dissolve starch completely in water it needs to be heated; when it cools down too much the starch solution forms a thermo-irreversible gel. Amylomaltases (EC 2.4.1.25) are enzymes that transfer a segment of an alpha-1,4-D-glucan to a new 4-position in an acceptor, which may be glucose or another alpha-1,4-D-glucan. Acting upon starch, amylomaltases can produce cycloamylose or a thermoreversible starch gel, both of which are of commercial interest.

Improved oligosaccharide synthesis by protein engineering of beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus.

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type beta-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type beta-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95 degrees C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as low as 10%, only trisaccharides were synthesized by CelB wild-type. The beta-mannosidase BmnA from P. furiosus showed both beta-glucosidase and beta-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating beta-glycosidases in oligosaccharide synthesis from lactose.

Activity and stability of hyperthermophilic enzymes: a comparative study on two archaeal beta-glycosidases.

S beta gly and CelB are well-studied hyperthermophilic glycosyl hydrolases, isolated from the Archaea Sulfolobus solfataricus and Pyrococcus furiosus, respectively. Previous studies revealed that the two enzymes are phylogenetically related; they are very active and stable at high temperatures, and their overall three-dimensional structure is very well conserved. To acquire insight in the molecular determinants of thermostability and thermoactivity of these enzymes, we have performed a detailed comparison, under identical conditions, of enzymological and biochemical parameters of S beta gly and CelB, and we have probed the basis of their stability by perturbations induced by temperature, pH, ionic strength, and detergents. The major result of the present study is that, although the two enzymes are remarkably similar with respect to kinetic parameters, substrate specificity, and reaction mechanism, they are strikingly different in stability to the different physical or chemical perturbations induced. These results provide useful information for the design of further experiments aimed at understanding the structure-function relationships in these enzymes.

Comparative structural analysis and substrate specificity engineering of the hyperthermostable beta-glucosidase CelB from Pyrococcus furiosus.

The substrate specificity of the beta-glucosidase (CelB) from the hyperthermophilic archaeon Pyrococcus furiosus, a family 1 glycosyl hydrolase, has been studied at a molecular level. Following crystallization and X-ray diffraction of this enzyme, a 3.3 A resolution structural model has been obtained by molecular replacement. CelB shows a homo-tetramer configuration, with subunits having a typical (betaalpha)(8)-barrel fold. Its active site has been compared to the one of the previously determined 6-phospho-beta-glycosidase (LacG) from the mesophilic bacterium Lactococcus lactis. The overall design of the substrate binding pocket is very well conserved, with the exception of three residues that have been identified as a phosphate binding site in LacG. To verify the structural model and alter its substrate specificity, these three residues have been introduced at the corresponding positions in CelB (E417S, M424K, F426Y) in different combinations: single, double, and triple mutants. Characterization of the purified mutant CelB enzyme revealed that F426Y resulted in an increased affinity for galactosides, whereas M424K gave rise to a shifted pH optimum (from 5.0 to 6.0). Analysis of E417S revealed a 5-fold and a 3-fold increase of the efficiency of hydrolyzing o-nitrophenol-beta-D-galactopyranoside-6-phosphate, in the single and triple mutants, respectively. In contrast, their activity on nonphosphorylated sugars was largely reduced (30-300-fold). The residue at position E417 in CelB seems to be the determining factor for the difference in substrate specificity between the two types of family 1 glycosidases.

Improving low-temperature catalysis in the hyperthermostable Pyrococcus furiosus beta-glucosidase CelB by directed evolution.

The beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus (CelB) is the most thermostable and thermoactive family 1 glycosylhydrolase described to date. To obtain more insight in the molecular determinants of adaptations to high temperatures and study the possibility of optimizing low-temperature activity of a hyperthermostable enzyme, we generated a library of random CelB mutants in Escherichia coli. This library was screened for increased activity on p-nitrophenyl-beta-D-glucopyranoside at room temperature. Multiple CelB variants were identified with up to 3-fold increased rates of hydrolysis of this aryl glucoside, and 10 of them were characterized in detail. Amino acid substitutions were identified in the active-site region, at subunit interfaces, at the enzyme surface, and buried in the interior of the monomers. Characterization of the mutants revealed that the increase in low-temperature activity was achieved in different ways, including altered substrate specificity and increased flexibility by an apparent overall destabilization of the enzyme. Kinetic characterization of the active-site mutants showed that in all cases the catalytic efficiency at 20 degrees C on p-nitrophenyl-beta-D-glucose, as well as on the disaccharide cellobiose, was increased up to 2-fold. In most cases, this was achieved at the expense of beta-galactosidase activity at 20 degrees C and total catalytic efficiency at 90 degrees C. Substrate specificity was found to be affected by many of the observed amino acid substitutions, of which only some are located in the vicinity of the active site. The largest effect on substrate specificity was observed with the CelB variant N415S that showed a 7.5-fold increase in the ratio of p-nitrophenyl-beta-D-glucopyranoside/p-nitrophenyl-beta-D-galactopyra noside hydrolysis. This asparagine at position 415 is predicted to interact with active-site residues that stabilize the hydroxyl group at the C4 position of the substrate, the conformation of which is equatorial in glucose-containing substrates and axial in galactose-containing substrates.

Alpha-frequency rhythms desynchronize over long cortical distances: a modeling study.

Neocortical networks of excitatory and inhibitory neurons can display alpha(a)-frequency rhythms when an animal is in a resting or unfocused state. Unlike some gamma- and beta-frequency rhythms, experimental observations in cats have shown that these alpha-frequency rhythms need not synchronize over long cortical distances. Here, we develop a network model of synaptically coupled excitatory and inhibitory cells to study this asynchrony. The cells of the local circuit are modeled on the neurons found in layer V of the neocortex where alpha-frequency rhythms are thought to originate. Cortical distance is represented by a pair of local circuits coupled with a delay in synaptic propagation. Mathematical analysis of this model reveals that the h and T currents present in layer V pyramidal (excitatory) cells not only produce and regulate the alpha-frequency rhythm but also lead to the occurrence of spatial asynchrony. In particular, these inward currents cause excitation and inhibition to have nonintuitive effects in the network, with excitation delaying and inhibition advancing the firing time of cells; these reversed effects create the asynchrony. Moreover, increased excitatory to excitatory connections can lead to further desynchronization. However, the local rhythms have the property that, in the absence of excitatory to excitatory connections, if the participating cells are brought close to synchrony (for example, by common input), they will remain close to synchrony for a substantial time.

Congenital coronary arteriovenous fistula associated with atrioventricular valvular regurgitation in an octogenarian.

An arteriovenous fistula originating from halfway down the circumflex coronary artery, draining into the coronary sinus was first diagnosed in a patient of 81. For 10 years, she had had dyspnea on exertion, mild mitral regurgitation and permanent ventricular pacing for symptomatic bradyarrhythmia. She was treated medically and despite her age was offered transcatheter balloon embolization, but she refused to undergo such intervention.