In vitro digestibility of commercial and experimental isomalto-oligosaccharides.

Isomalto-oligosaccharides (IMO) significantly contribute to the global oligosaccharide market. IMO are linear α-(1 â†’ 6) linked oligosaccharides with isomaltotriose as the representative trisaccharide. Commercial IMO preparations ypically also contain panose-series oligosaccharides as a major component. In humans, IMO are partially digestible but the digestibility of specific components of commerical IMO preparations remains unknown. This study aimed to compare the in vitro digestibility of reference compounds, experimental α-gluco-oligosaccharides and commercial IMO. Experimental α-gluco-oligosaccharides were synthesized with the recombinant dextransucrase DsrM. Two in vitro digestion methods were used, a reference method matching the AOAC method for dietary fibre, and a protocol that uses brush border glycosyl hydrolases from the rat intestine. The α-gluco-oligosaccharides patterns after hydrolysis remain were analyzed by high performance anion exchange chromatography coupled to pulsed amperometric detection. Panose-series oligosaccharides were hydrolysed more rapidly by amylase and amyloglucosidase when compared to hydrolysis by rat intestinal enzymes. The rate of hydrolysis by rat intestinal enzymes decreased in the order panose > isomaltose, kojibiose or nigerose. Hydrolysis of panose-series oligosaccharides but not the hydrolysis of isomalto-oligosaccharides was dependent on the degree of polymerization. Qualitative analysis of oligosaccharides remaining after hydrolysis indicated that rat small intestinal enzymes hydrolyse their substrates from the non-reducing end. Taken together, results inform on the modification or optimization of current production processes for IMO to obtain tailored oligosaccharide preparations with reduced digestibility and an increased content of dietary fibre.

Suture length to wound length ratio in 175 small animal abdominal midline closures.

Experimental and human studies have reported the advantages of a suture length to wound length (SL:WL) ratio greater than 4:1 in midline abdominal closure. This is achieved when the tissue bite (TB) is equal to or larger than the stitch interval (SI). Although TB and SI values are recommended in some textbooks, SL:WL ratios are rarely reported in veterinary textbooks. Additionally, no clinical data regarding these parameters could be found in small animals. Therefore, the aim of this study was to evaluate the SL:WL ratio of midline laparotomy closure in dogs and cats performed by surgeons with different levels of expertise and to compare the findings with current textbook recommendations. Midline laparotomy incisions of 100 dogs and 75 cats were closed in continuous pattern by diplomates and residents of both the European College of Veterinary Surgeons (ECVS) and the European College of Animal Reproduction (ECAR). The mean SL:WL ratio was 2.5 ± 0.7:1. The surgeons´ level of experience and the species and body weights of the animals did not have any significant influence on the SL:WL ratio. A moderate negative correlation was observed between the mean SI to mean TB (SI:TB) ratio and the SL:WL ratio. In this study, the mean SI matched the textbook recommendations both in feline and canine species, whereas the TB in cats was different. In this study, the SL:WL ratio was less than 4:1 without apparent complications. Because of the low prevalence of incisional hernia in dogs and cats larger studies are necessary to evaluate clinical significance of the presented data.

Effect of acceptor carbohydrates on oligosaccharide and polysaccharide synthesis by dextransucrase DsrM from Weissella cibaria.

The digestibility of isomalto-oligosaccharides (IMO) as well as their metabolism by gut microbiota depends on the degree of polymerization and the ratio of α-(1→4) to α-(1→6) linkages. Both parameters are influenced by the method of production. Commercial IMO are produced by transglycosylation of starch hydrolysates, or by transglycosylation with dextransucrase and sucrose as glucosyl-donor and maltose as glucosyl-acceptor. This study aimed to quantitatively and qualitatively assess the acceptor reaction with dextransucrase. α-Glucans were selected by systematic variation of degree of polymerization and linkage type; the dextransucrase DsrM from Weissella cibaria 10M was used as biocatalyst. The efficiency of α-glucans as acceptor carbohydrates decreased in the order DP2>DP3>DP1; among disaccharides, the efficiency decreased in the order α-(1→6)>α-(1→4)>α-(1→3); the α-(1→2) linked kojibiose did not support oligosaccharide formation. Equimolar addition of efficient acceptor molecules and sucrose shifted the dextransucrase reaction to oligosaccharides as virtually exclusive product. DsrM readily extended a commercial IMO preparation by adding α-(1→6)-linked glucose moieties. Conversion of commercial IMO by dextransucrase reduced their in vitro digestibility as analyzed by two different protocols. This study facilitates the synthesis of oligosaccharides produced in the acceptor reaction with dextransucrase with controlled yields and degree of polymerization, and hence with optimal functional properties in food applications.

The changing fate of a secretory glycoprotein in developing maize endosperm.

Zeins are the major storage proteins in maize (Zea mays) endosperm, and their accumulation in zein bodies derived from the endoplasmic reticulum is well characterized. In contrast, relatively little is known about post-Golgi compartments or the trafficking of vacuolar proteins in maize endosperm, specifically the presence of globulins in structures resembling protein storage vacuoles that appear in early to mid-stage seed development. We investigated this pathway by expressing and analyzing a recombinant reporter glycoprotein during endosperm maturation, using a combination of microscopy and sensitive glycopeptide analysis. Specific N-glycan acceptor sites on the protein were followed through the stages of grain development, revealing a shift from predominantly paucimannosidic vacuolar glycoforms to predominantly trimmed glycan structures lacking fucose. This was accompanied by a change in the main subcellular localization of the protein from large protein storage vacuole-like post-Golgi organelles to the endoplasmic reticulum and zein bodies. The endogenous storage proteins corn alpha-globulin and corn legumin-1 showed a similar spatiotemporal profile both in transgenic plants expressing the reporter glycoprotein and in wild-type plants. This indicates that the shift of the intracellular trafficking route, as observed with our reporter glycoprotein, may be a common strategy in maize seed development.

Exploring the ESCRTing machinery in eukaryotes.

The profile of protein sorting into multivesicular bodies (MVBs) has risen recently with the identification of three heteromeric complexes known as ESCRT-I,-II,-III (Endosomal Sorting Complex Required for Transport). Genetic analyses in yeast have identified up to 15 soluble class E VPS (vacuolar protein sorting) proteins that have been assigned to the ESCRT machinery and function in cargo recognition and sorting, complex assembly, vesicle formation and dissociation. Despite their functional importance in yeast and mammalian cells, little is known about their presence and function in other organisms including plants. We have made use of the fully sequenced genomes of Arabidopsis thaliana and Oryza sativa, Drosophila melanogaster and Caenorhabditis elegans to explore the identity, structural characteristics and phylogenetic relationships of proteins assigned to the ESCRT machinery.