Modeling and biochemical analysis of the activity of antibiofilm agent Dispersin B.

Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix (PGA), which is a linear polymer of beta(1,6)-linked N-acetylglucosamine (GlcNAc) residues. Dispersin B (DspB), a soluble glycoside hydrolase produced by the periodontal pathogen Actinobacillus actinomycetemcomitans degrades PGA. The enzyme DspB is an alpha/beta TIM-barrel protein and belongs to family 20 glycosyl hydrolases members. The enzyme activity of DspB with regard to its substrate specificity towards beta(1,6)-linked GlcNAc polymers and its endo/exo character was investigated through ligand docking and the hydrolysis of synthetic oligosaccharides. Ligand docking analysis suggested that beta(1,6)-linked GlcNAc oligosaccharide bound to the active site better that beta(1,4)-linked GlcNAc oligosaccharide. Our combined results indicate that DspB is an exo-acting enzyme that hydrolyzes beta(1,6)-linked N-acetylglucosamine oligomers.

Scope and limitation of the application of the (methoxydimethyl)methyl group in the synthesis of 2'-O-, 6'-O- and 2',6'-di-O-(alpha-L-arabinofuranosyl)-beta-D-galactopyranosyl- (1-->6)-D-galactoses.

For the characterisation of the anticipated epitope of an arabinogalactan, isolated from the extract of Echinacea purpurea, the trisaccharide alpha-L-Araf-(1-->2)-beta-D-Galp- (1-->6)-D-Gal was synthesized. The easily available 3,4-O-isopropylidene-6-O-(methoxydimethyl)methyl-beta-D-galactopyr anosyl- (1-->6)-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranose having the OH-2' free served as aglycone upon direct arabinosylation at the 2' position under Helferich conditions. The formed HgBr2 cleaved the acid sensitive protecting group at position 6', but under basic conditions the desired, fully protected trisaccharide, or its symmetrical dimerization derivative linked 6'- to 6'-position via an isopropylidene bridge, could be obtained. An alternative route involved arabinosylation of a hepta-O-acetylated digalactose with free OH-2'. Two other oligosaccharides, namely, alpha-L-Araf-(1-->6)-beta-D-Galp-(1-->6)-D-Gal and (alpha-L-Araf)2-(1-->2,6)-beta-D-Galp-(1-->6)-D-Gal were also synthesized and characterised.

Synthesis of chromogenic substrates of alpha-amylases on a cyclodextrin basis.

One-pot acetylation and subsequent partial acetolysis of alpha-, beta- and gamma-cyclodextrins resulted in crystalline peracetylated malto-hexaose, -heptaose, and -octaose, respectively. Prolonged acetolysis of beta-cyclodextrin gave a mixture of acetylated maltooligosaccharides, from which peracetylated malto-triose, -tetraose, and -pentaose were isolated. The acetylated oligosaccharides were converted into alpha-acetobromo derivatives, and then transformed into 4-nitrophenyl and 2-chloro-4-nitrophenyl beta-glycosides. From the 4-nitrophenyl glycosides 4,6-O-benzylidene derivatives were prepared, which were used together with the free glycosides as substrates of porcine pancreatic alpha-amylase.

Synthesis of the alpha-L-Araf-(1-->2)-beta-D-Galp-(1-->6)-beta-D-Galp-(1-->6)-[alpha-L-Araf-(1-->2)]-beta-D-Galp-(1-->6)-D-Gal hexasaccharide as a possible repeating unit of the cell-cultured exudates of Echinacea purpurea arabinogalactan.

For the characterization of the supposed epitope of an arabinogalactan, isolated from the extract of the cell-cultured Echinacea purpurea, the title hexasaccharide was synthesized. The whole synthetic route was based on the 6-O-(methoxydimethyl)methyl ether (MIP) protecting group strategy. 2-O-Benzyl-3,4-O-isopropylidene-6-O-(methoxydimethyl)methyl-beta-D-galactopyranosyl-(1-->6)-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranose was used to prepare the desired glycosyl donor and glycosyl acceptor both carrying a persistent O-benzyl group at position 2'. Reaction of the digalactose donor and the digalactose acceptor resulted in a beta-(1-->6)-linked galactose-containing tetrasaccharide in which OH-2' and OH-2"' were substituted with benzyl groups. Hydrogenolytic removal of the benzyl groups of the tetragalactose compound gave the diol aglycon which was diarabinosylated in one step to furnish the protected target compound, whose deprotection led to the title hexasaccharide. All of the synthesized compounds were characterized by 1H and 13C NMR spectra, as well as by MALDI-TOF mass-spectrometry measurements.

Quantitative light microscopic study on the distribution of Kupffer cells during chemical hepatocarcinogenesis in the rat.

The quantitative distribution of Kupffer cells was measured in the liver of Fischer-344 male rats during the course of chemical hepatocarcinogenesis according to the Solt-Farber model, i.e. an initiating agent, a selective mitogenic inhibitor and a strong proliferation stimulus were applied subsequently. Pre-neoplastic and neoplastic lesions were followed up to 17 months. Kupffer cells were detected by the phagocyted carrageenan stained with toluidine blue and were counted using the ocular square in the light microscope. In the non-transformed areas of the liver of treated rats the number of Kupffer cells was generally different from the values of untreated controls. The altered cell foci contained fewer Kupffer cells than the normal untreated rats and the non-transformed liver areas, while in hepatocellular carcinomas there was a high reduction in the number of detectable Kupffer cells. The number of Kupffer cells was variable around and within neoplastic nodules. While Kupffer cells do not follow the neoplastic proliferation of hepatocytes during hepatocarcinogenesis, it is possible that humoral factors are produced by transformed hepatocytes against Kupffer cells.

A new mixed acetal-type substitution pattern for alpha-cyclodextrin. Preparation of hexakis (3-O-benzyl)- alpha-cyclodextrin.

alpha-CD was converted into hexakis[2,6-di-O-(methoxydimethyl)methyl]-alpha-CD by a proton-catalyzed reaction with 2-methoxypropene. Subsequent benzylation under Brimacombe conditions resulted in the fully protected compound, from which the acid-sensitive acetal groups were removed to obtain hexakis(3-O-benzyl)-alpha-cyclodextrin. The structure of all of the compounds synthesized was confirmed by 13C J-ECHO, COSY, HETCOR and HMBC NMR measurements.