Glycosidase mechanisms

Insights into glycosidase mechanisms have come from X-ray crystallographic studies on complexes with substrate analogs and inhibitors, representing all the intermediate species along the reaction coordinate. Site-directed mutagenesis continues to play a significant role in understanding mechanisms, but is also proving important in generating glycosidases of modified mechanism or specificity.     

Glutamate dehydrogenase activity in lactobacilli and the use of glutamate dehydrogenase-producing adjunct Lactobacillus spp. cultures in the manufacture of cheddar cheese

AIMS: The study was undertaken to investigate the occurrence of glutamate dehydrogenase activity in different species of lactobacilli, and to determine, in a series of cheese-making trials, the effects of glutamate dehydrogenase-producing adjunct cultures on sensory attribute development during the maturation of cheddar cheese. METHODS AND RESULTS: The presence of dehydrogenase activity with glutamate as substrate was monitored in cell lysates of >100 strains from 30 different species of lactobacilli using a qualitative colorimetric plate screening assay. Activity was detectable in 25 of the 29 representative species obtained from culture collections and in 12 of the 13 non-starter species isolated from cheese. There were pronounced interspecies and strain differences in the occurrence, level and pyridine nucleotide specificity of the glutamate dehydrogenase activity detected. Among the non-starter lactobacilli the highest frequency of enzyme occurrence and activity was detected in the Lactobacillus plantarum isolates. The establishment of glutamate dehydrogenase-producing adjunct strains in the predominant population of lactobacilli in the cheese curd affected the formation of a number of volatile compounds in ripening cheddar cheese, while the presence of Lact. plantarum strains, in particular, was associated with an intensification and acceleration of aroma and flavour development during the maturation period. CONCLUSIONS: Glutamate dehydrogenase formation by lactobacilli is a strain-dependent metabolic attribute, and adjunct cultures expressing the activity that are able to proliferate during cheese ripening have a positive impact on the rate of development and the intensity of cheddar cheese aroma and flavour development. SIGNIFICANCE AND IMPACT OF THE STUDY: It has been demonstrated that some strains of glutamate dehydrogenase-producing lactobacilli have potential use as adjunct cultures to accelerate and intensify aroma and flavour formation during the manufacture of cheddar and, by analogy, other similar varieties of cheese. The importance of phenotypic discriminative monitoring of the dominant lactobacilli present during ripening to confirm adjunct establishment and population complexity was highlighted as was the requirement to establish the metabolic attributes of the non-starter population in uninoculated control cheeses in comparative trials.     

Fluorescently tagged iminoalditol glycosidase inhibitors as novel biological probes and diagnostics

1,5-Dideoxy-1,5-imino-D-glucitol, the corresponding D-manno and L-ido epimers as well as the powerful beta-glucosidase inhibitor isofagomine were N-alkylated with di-, tri-, as well as tetraethylene glycol derived straight chain spacer arms by a set of simple standard procedures. The terminal functional groups of the spacer arms, primary amines, were employed to introduce fluorescent dansyl moieties. Resulting derivatives showed glycosidase inhibitory activities comparable to those of the parent compounds'.     

Multiple, non-allelic, intein-coding sequences in eukaryotic RNA polymerase genes

Background  

Inteins are self-splicing protein elements. They are translated as inserts within host proteins that excise themselves and ligate the flanking portions of the host protein (exteins) with a peptide bond. They are encoded as in-frame insertions within the genes for the host proteins. Inteins are found in all three domains of life and in viruses, but have a very sporadic distribution. Only a small number of intein coding sequences have been identified in eukaryotic nuclear genes, and all of these are from ascomycete or basidiomycete fungi.     

A novel chemogenomics analysis of G protein-coupled receptors (GPCRs) and their ligands: a potential strategy for receptor de-orphanization

Background  

G protein-coupled receptors (GPCRs) represent a family of well-characterized drug targets with significant therapeutic value. Phylogenetic classifications may help to understand the characteristics of individual GPCRs and their subtypes. Previous phylogenetic classifications were all based on the sequences of receptors, adding only minor information about the ligand binding properties of the receptors. In this work, we compare a sequence-based classification of receptors to a ligand-based classification of the same group of receptors, and evaluate the potential to use sequence relatedness as a predictor for ligand interactions thus aiding the quest for ligands of orphan receptors.     

Controlled delivery of antisense oligodeoxynucleotide from cationically modified phosphorylcholine polymer films

Antisense strategy is a promising approach for the prevention of in-stent restenosis if therapeutic agents such as antisense oligodeoxynucleotides (AS-ODNs) can be successfully delivered to the implant site. Optimizing the routes and conditions for controlled loading and release of therapeutic agents from a biocompatible polymer coating is still required. In this study, phosphorylcholine (PC) polymer films bearing different cationic charge densities were deposited onto smooth silicon substrates. The thickness of these films was determined by spectroscopic ellipsometry (SE). Human c-myc AS-ODNs were incorporated into the PC polymer films by immersion in concentrated AS-ODN solution and eluted into PBS under physiological conditions. The elution profile was monitored by UV spectrometry and gel electrophoresis. Cellular uptake of the eluted AS-ODN into vascular smooth muscle cells (VSMCs) was evaluated by fluorescence microscopy. The results showed that ODN loading capacities increased with film thickness and were also strongly dependent on the cationic charge density. AS-ODN release was characterized by a slight initial burst in the first half hour followed by a period of sustained release up to 8 days. Gel electrophoresis demonstrated DNA integrity, and different transfection efficiencies were observed when the eluted ODNs were transfected into VSMCs. These results demonstrated that cationically modified PC polymers are capable of delivery of antisense ODNs in a controlled manner and that they are well suited for specific biomedical devices such as DNA-eluting stents.     

Critical role for peptide YY in protein-mediated satiation and body-weight regulation

Dietary protein enhances satiety and promotes weight loss, but the mechanisms by which appetite is affected remain unclear. We investigated the role of gut hormones, key regulators of ingestive behavior, in mediating the satiating effects of different macronutrients. In normal-weight and obese human subjects, high-protein intake induced the greatest release of the anorectic hormone peptide YY (PYY) and the most pronounced satiety. Long-term augmentation of dietary protein in mice increased plasma PYY levels, decreased food intake, and reduced adiposity. To directly determine the role of PYY in mediating the satiating effects of protein, we generated Pyy null mice, which were selectively resistant to the satiating and weight-reducing effects of protein and developed marked obesity that was reversed by exogenous PYY treatment. Our findings suggest that modulating the release of endogenous satiety factors, such as PYY, through alteration of specific diet constituents could provide a rational therapy for obesity.     

1-Deoxynojirimycins with dansyl capped N-substituents as probes for Morbus Gaucher affected cell lines

Cyclization by double reductive amination of d-xylo-hexos-5-ulose with methyl 6-aminohexanoate gave (methoxycarbonyl)pentyl-1-deoxynojirimycin. Reaction of the terminal carboxylic acid with N-dansyl-1,6-diaminohexane provided the corresponding chain-extended fluorescent derivative. By reaction with bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Both compounds are strong inhibitors of d-glucosidases and could also be shown to distinctly improve, at sub-inhibitory concentrations, the activity of β-glucocerebrosidase in a Gaucher fibroblast (N370S) cell-line through chaperoning of the enzyme to the lysosome.     

Syntheses of p-nitrophenyl 3- and 4-thio-β-D-glycopyranosides

Thioglycosides have proved to be useful, enzymatically stable analogs of glycosides for structural and mechanistic studies and their synthesis is considerably simplified through the use of thioglycoligases. As part of an investigation into the use of thioglycosides as potential pharmacological chaperones, and as components of glycoproteins and glycolipids, the syntheses of p-nitrophenyl 3-thio-β-d-galactopyranoside, phenyl 1,4-dithio-β-d-glucopyranoside, p-nitrophenyl 4-thio-β-d-mannopyranoside and p-nitrophenyl 2-acetamido-2-deoxy-4-thio-β-d-mannopyranoside are described.     

Effects of hypothyroidism on myosin heavy chain composition and fibre types of fast skeletal muscles in a small marsupial, Antechinus flavipes

Effects of drug-induced hypothyroidism on myosin heavy chain (MyHC) content and fibre types of fast skeletal muscles were studied in a small marsupial, Antechinus flavipes. SDS-PAGE of MyHCs from the tibialis anterior and gastrocnemius revealed four isoforms, 2B, 2X, 2A and slow, in that order of decreasing abundance. After 5 weeks treatment with methimazole, the functionally fastest 2B MyHC significantly decreased, while 2X, 2A and slow MyHCs increased. Immunohistochemistry using monospecific antibodies to each of the four MyHCs revealed decreased 2b and 2x fibres, and increased 2a and hybrid fibres co-expressing two or three MyHCs. In the normally homogeneously fast superficial regions of these muscles, evenly distributed slow-staining fibres appeared, resembling the distribution of slow primary myotubes in fast muscles during development. Hybrid fibres containing 2A and slow MyHCs were virtually absent. These results are more detailed but broadly similar to the earlier studies on eutherians. We hypothesize that hypothyroidism essentially reverses the effects of thyroid hormone on MyHC gene expression of muscle fibres during myogenesis, which differ according to the developmental origin of the fibre: it induces slow MyHC expression in 2b fibres derived from fast primary myotubes, and shifts fast MyHC expression in fibres of secondary origin towards 2A, but not slow, MyHC.