Метод проектів у процесі музично-виконавської підготовки майбутнього вчителя музики

(uk) У статті визначається сутність інтегрованого методу художньо-творчих проектів, розкривається його специфіка у процесі музично-виконавської підготовки майбутніх учителів музики.(ru) В статье определяется сущность интегрированного метода художественно-творческих проэктов, раскрывается его специфика в процессе музыкально-исполнительской подготовки будущих учителей музыки

Structures of a human blood group glycosyltransferase in complex with a photo-activatable UDP-Gal derivative reveal two different binding conformations.

International audienceGlycosyltransferases (GTs) catalyse the sequential addition of monosaccharides to specific acceptor molecules and play major roles in key biological processes. GTs are classified into two main families depending on the inverted or retained stereochemistry of the glycosidic bond formed during the reaction. While the mechanism of inverting enzymes is well characterized, the precise nature of retaining GTs is still a matter of much debate. In an attempt to clarify this issue, studies were initiated to identify reaction-intermediate states by using a crystallographic approach based on caged substrates. In this paper, two distinct structures of AA(Gly)B, a dual-specificity blood group synthase, are described in complex with a UDP-galactose derivative in which the O6'' atom is protected by a 2-nitrobenzyl group. The distinct conformations of the caged substrate in both structures of the enzyme illustrate the highly dynamic nature of its active site. An attempt was also made to photolyse the caged compound at low temperature, which unfortunately is not possible without damaging the uracil group as well. These results pave the way for kinetic crystallography experiments aiming at trapping and characterizing reaction-intermediate states in the mechanism of enzymatic glycosyl transfer

Structural, biochemical and in vivo investigations of the threonine synthase from Mycobacterium tuberculosis

Threonine biosynthesis is a general feature of prokaryotes, eukaryotic microorganisms, and higher plants. Since mammals lack the appropriate synthetic machinery, instead obtaining the amino acid through their diet, the pathway is a potential focus for the development of novel antibiotics, antifungal agents, and herbicides. Threonine synthase (TS), a pyridoxal-5-phosphate-dependent enzyme, catalyzes the final step in the pathway, in which L-homoserine phosphate and water are converted into threonine and inorganic phosphate. In the present publication, we report structural and functional studies of Mycobacterium tuberculosis TS, the product of the rv1295 (thrC) gene. The structure gives new insights into the catalytic mechanism of TSs in general, specifically by suggesting the direct involvement of the phosphate moiety of the cofactor, rather than the inorganic phosphate product, in transferring a proton from C4' to C-gamma in the formation of the alpha beta-unsaturated aldimine. It further provides a basis for understanding why this enzyme has a higher pH optimum than has been reported elsewhere for TSs and gives rise to the prediction that the equivalent enzyme from Thermus thermophilus will exhibit similar behavior. A deletion of the relevant gene generated a strain of M. tuberculosis that requires threonine for growth, such auxotrophic strains are frequently attenuated in vivo, indicating that TS is a potential drug target in this organism

Thiogalactopyranosides are resistant to hydrolysis by \u3b1-galactosidases

Fluorescently tagged glycosides containing terminal \u3b1(1\u21923) and \u3b1(1\u21924)-linked thiogalactopyranosides have been prepared and tested for resistance to hydrolysis by \u3b1-galactosidases. Eight fluorescent glycosides containing either galactose or 5-thiogalactose as the terminal sugar were enzymatically synthesized using galactosyltransferases, with lactosyl glycosides as acceptors and UDP-galactose or UDP-5\u2032-thiogalactose, respectively, as donors. The glycosides were incubated with human \u3b1-galactosidase A (CAZy family GH27, a retaining glycosidase), Bacteroides fragilis \u3b1-1,3-galactosidase (GH110, an inverting glycosidase), or homogenates of MCF-7 human breast cancer cells or NG108-15 rat glioma cells. Substrate hydrolysis was monitored by capillary electrophoresis with fluorescence detection. All compounds containing terminal O-galactose were readily degraded. Their 5-thiogalactose counterparts were resistant to hydrolysis by human \u3b1-galactosidase A and the enzymes present in the cell extracts. B. fragilis \u3b1-1,3-galactosidase hydrolyzed both thio- and O-galactoside substrates; however, the thiogalactosides were hydrolyzed at only 1\u20133\u2009% of the rate of O-galactosides. The hydrolytic resistance of 5-thiogalactose was also confirmed by an in vivo study using cells in culture. The results suggest that 5-thiogalactosides may be useful tools for the study of anabolic pathways in cell extracts or in single cells.Peer reviewed: YesNRC publication: Ye

Neoglycolipids for Prolonging the Effects of Peptides: Self-Assembling Glucagon-like Peptide 1 Analogues with Albumin Binding Properties and Potent in Vivo Efficacy

Novel principles for optimizing the properties of peptide-based drugs are needed in order to leverage their full pharmacological potential. We present the design, synthesis, and evaluation of a library of neoglycolipidated glucagon-like peptide 1 (GLP-1) analogues, which are valuable drug candidates for treatment of type 2 diabetes and obesity. Neoglycolipidation of GLP-1 balanced the lipophilicity, directed formation of soluble oligomers, and mediated albumin binding. Moreover, neoglycolipidation did not compromise bioactivity, as in vitro potency of neoglycolipidated GLP-1 analogues was maintained or even improved compared to native GLP-1. This translated into pronounced in vivo efficacy in terms of both decreased acute food intake and improved glucose homeostasis in mice. Thus, we propose neoglycolipidation as a novel, general method for modulating the properties of therapeutic peptides

Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum

Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from alpha-D-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of alpha-D-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming beta-glucosyl disaccharides with beta-(1-->4)- regioselectivity from five monosaccharides as well as branched beta-glucosyl trisaccharides with beta-(1-->4)-regioselectivity from three (1-->6)-linked disaccharides. CtCDP showed strict beta-(1-->4)-regioselectivity and catalysed linear chain extension of the three beta-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two beta-glucosyl oligosaccharide product series to represent novel compounds, i.e. beta-D-glucopyranosyl-[(1-->4)-beta-D-glucopyranosyl](n)-(1-->2)-D-gluco pyranose, and beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranosyl](n)-(1-->3)-D-glucop yranose (n = 1-7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the Cl hydroxyl of beta-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of Cl substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637-Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using alpha-D-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides