Mechanism of chitosanase-oligosaccharide interaction: subsite structure of Streptomyces sp. N174 chitosanase and the role of Asp57 carboxylate.

We have investigated the mechanism of the interaction of Streptomyces sp. N174 chitosanase with glucosamine hexasaccharide [(GlcN)(6)] by site-directed mutagenesis, thermal unfolding, and (GlcN)(6) digestion experiments, followed by theoretical calculations. From the energy-minimized model of the chitosanase-(GlcN)(6) complex structure (Marcotte et al., 1996), Asp57, which is present in all known chitosanases, was proposed to be one of the amino acid residues that interacts with the oligosaccharide substrate. The chitosanase gene was mutated at Asp57 to Asn (D57N) and Ala (D57A), and the relative activities of the mutated chitosanases were found to be 72 and 0.5% of that of the wild type, respectively. The increase in the transition temperature of thermal unfolding (T(m)), usually observed upon the addition of (GlcN)(n) to chitosanase mutants unaffected in terms of substrate binding, was considerably suppressed in the D57A mutant. These data suggest that Asp57 is important for substrate binding. The experimental time-courses of [(GlcN)(6)] degradation were analyzed by a theoretical model in order to obtain the binding free energy values of the individual subsites of the chitosanases. A (-3, -2, -1, +1, +2, +3) subsite model agreed best with the experimental data. This analysis also indicated that the mutation of Asp57 affects substrate affinity at subsite (-2), suggesting that Asp57 most likely participates in the substrate binding at this subsite.

[Nursing care of elderly persons: reactions to the use of a security blanket to prevent falling out of bed]. / Soins infirmiers aux personnes âgées: réactions à l'utilisation de la couverture de sécurité dans la prévention des chutes de lit.

This article presents the results of the first part of a survey aiming at assessing the chances for adoption and use of the safety blanket, a new device preventing the falls from the beds. In this part, the resarchers wanted to know how the caretakers reacted to the use of this material. Thirty four people with five beneficiaries among them, nine family members, fifteen contributors and five managers, interacting in the context of a care unit for elderly people of a hospital centre were interviewed. The data of the interviews were analysed according to a six step procedure: listening to the interviews and reading the descriptions; deriving the significant statements, analysing and reformulating the meaning of the statements; regrouping the signification units under more global themes; gathering the analysis results and describing exhaustively the studied phenomenon; validating the exhaustive description. As a whole, the reactions recorded were positive and indicate that the safety blanket has big chances to be adopted by the healthcare units.

A common molecular signature unifies the chitosanases belonging to families 46 and 80 of glycoside hydrolases.

The 3D structure-oriented alignment of the primary sequences of fourteen chitosanases, mainly of bacterial origin and belonging to families 46 and 80 of glycoside hydrolases, resulted in the identification of the following pattern common to all these enzymes: E-[DNQ]-x(8,17)-Y-x(7)-D-x-[RD]-[GP]-x-[TS]-x(3)-[AIVFLY]-G- x(5,11)-D. This pattern is proposed as the molecular signature of the chitosanases from families 46 and 80. It includes several amino acids essential for enzyme activity and (or) stability as shown by site-directed mutagenesis studies on the chitosanase from Streptomyces sp. N174. In particular, it includes two carboxylic residues directly involved in catalysis. We suggest that there is a continuum of sequence similarity between all the analyzed chitosanases, and that all these enzymes should probably be classified in one family.

Theoretical calculation of pKa reveals an important role of Arg205 in the activity and stability of Streptomyces sp. N174 chitosanase.

Based on the crystal structure of chitosanase from Streptomyces sp. N174, we have calculated theoretical pK(a) values of the ionizable groups of this protein using a combination of the boundary element method and continuum electrostatics. The pK(a) value obtained for Arg(205), which is located in the catalytic cleft, was abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. Chitosanases possessing mutations in this position (R205A, R205H, and R205Y), produced by Streptomyces lividans expression system, were found to have less than 0.3% of the activity of the wild type enzyme and to possess thermal stabilities 4-5 kcal/mol lower than that of the wild type protein. In the crystal structure, the Arg(205) side chain is in close proximity to the Asp(145) side chain (theoretical pK(a), -1.6), which is in turn close to the Arg(190) side chain (theoretical pK(a), 17.7). These theoretical pK(a) values are abnormal, suggesting that both of these residues may participate in the Arg(205) interaction network. Activity and stability experiments using Asp(145)- and Arg(190)-mutated chitosanases (D145A and R190A) provide experimental data supporting the hypothesis derived from the theoretical pK(a) data and prompt the conclusion that Arg(205) forms a strong interaction network with Asp(145) and Arg(190) that stabilizes the catalytic cleft.

Effects of gamma-hydroxybutyrate on ventral tegmental unit activity in the rat: considerations on rem sleep control.

The effect of gamma-hydroxybutyrate (GHB) administration on spontaneously active dopaminergic cells of the ventral tegmental area (VTA) was determined using extracellular single unit recordings in urethane-anesthetized rats. High doses (160-250 mg/kg, i.p.) of GHB reversibly decreased firing rate in 63.6% of the cells tested (n=11); remaining cells (36.4%) were unaffected. When the GHB receptor antagonist NCS-382 (10 mg/kg, i.p.) was co-administered with GHB at high doses, 50% of the cells became excited while remaining cells were unaffected. Of the 34 cells tested with GHB at low doses (10 mg/kg, i.p.), 21 (61.8%) changed their firing activity. Of these, 12 (57.1%) were excited, five (23.8%) were inhibited, and four (19.0%) were first excited then totally inhibited (E/Ipattern). Out of the three E/I cells tested, two resumed their firing activity after apomorphine (50 microgram/kg s.c.), showing that they were in a state of depolarization inactivation. When NCS-382 (10 mg/kg, i.p.) was co-administered with GHB at low doses, only two of the seven cells tested (28.6%) changed their firing activity, both with excitations. We conclude that only low doses of GHB selectively activate GHB receptors. Mechanisms by which low doses of GHB facilitate REM sleep are discussed.