Role of Rab9 GTPase in facilitating receptor recruitment by TIP47

Mannose 6-phosphate receptors (MPRs) deliver lysosomal hydrolases from the Golgi to endosomes and then return to the Golgi complex. TIP47 recognizes the cytoplasmic domains of MPRs and is required for endosome-to-Golgi transport. Here we show that TIP47 also bound directly to the Rab9 guanosine triphosphatase (GTPase) in its active, GTP-bound conformation. Moreover, Rab9 increased the affinity of TIP47 for its cargo. A functional Rab9 binding site was required for TIP47 stimulation of MPR transport in vivo. Thus, a cytosolic cargo selection device may be selectively recruited onto a specific organelle, and vesicle budding might be coupled to the presence of an active Rab GTPase.     

Reduction of fumonisin B1 in corn grits by single-screw extrusion

This study was designed to determine the efficacy of extrusion in reducing fumonisin B1 in corn flaking grits in the presence and absence of glucose. In addition, degradation products of fumonisin B1 during extrusion were identified and quantitated with a mass balance approach. Uncontaminated clean corn grits, grits spiked with 30 microg/g fumonisin B1, and grits fermented with Fusarium verticillioides M-2552 (40-50 microg/g fumonisin B1) were extruded in the presence and absence of glucose (10%, w/w) using a single-screw extruder. Extrusion decreased fumonisin B1 by 21-37%, whereas the same process with added glucose further decreased fumonisin B1 by 77-87%. LC-fluorescence and LC-MS showed that most fumonisin in the extruded samples without added glucose was the fumonisin B1 form, whereas the main degradation product in grits extruded with glucose was N-(deoxy- d-fructos-1-yl)fumonisin B1. The formation of hydrolyzed fumonisin B1 was not significant during extrusion. Results suggest that extrusion in the presence of glucose may reduce fumonisin B1 in corn grits significantly.     

Characterization and fate of black currant and bilberry flavonols in enzyme-aided processing

The fate of black currant ( Ribes nigrum L.) and bilberry ( Vaccinium myrtillus L.) flavonols in enzyme-aided processing was studied. The flavonols were quantified and characterized by high-performance liquid chromatography equipped with a diode array detector and an electrospray ionization mass spectrometer. A tentative identification for 14 black currant and 19 bilberry flavonols is presented representing 11 previously unpublished conjugates. For the first time in any berry, the presence of laricitrin conjugates is reported. The enzyme-aided processing affected the flavonol extractability, elevating the yield in juices and decreasing that in press residues. Importantly, no significant loss of the berry flavonols was observed during the experiments, although some hydrolysis of flavonol conjugates was recorded. To maximize the effect on flavonol extractability, higher enzyme dosages were needed for black currants than for bilberries. The data show that the flavonol extractability and hydrolysis are dependent on the texture of raw material, the glycosylation pattern of the conjugates, and the activity profile of the enzyme preparation.     

Determining species diversity of microfungal communities in forest tree roots by pure-culture isolation and DNA sequencing

Pure-culture isolation from roots was compared with transformation of total DNA from roots followed by sequence analysis of ITS 1/2 rDNA of representative clones as methods for determining the abundance and composition of microbiota in roots of Betula pendula, Fagus sylvatica, Larix decidua, Prunus serotina and Quercus petraea. The results from the two methods differed greatly, with no overlap between the taxa identified. Pure-culture isolation revealed greater species diversity (47 taxa), the most frequent fungi being Ascomycota, including Penicillium spp., Phialocephala fortinii, Pochonia bulbillosa, Sesquicillium candelabrum and Trichoderma spp. Transformation of total DNA and sequencing revealed less diversity (22 taxa), the most frequent taxa being Basidiomycota, including Coprinus fissolanatus and Mycena spp., and Ascomycota, including Podospora–Schizothecium spp., Helgardia anguioides and Microdochium sp. Communities characterized by either method showed slightly greater fungal diversity and less species dominance on F. sylvatica than on roots of other trees, whilst DNA sequencing showed least diversity and greatest species dominance on Q. petraea.     

Dose-dependent influence of commercial garlic (Allium sativum) on rats fed cholesterol-containing diet

The aim of this investigation was to evaluate the dose-dependent influence of commercial garlic on rats fed cholesterol-containing diets. It was found that commercial garlic contains high concentrations of dietary fibers, microelements, and total polyphenols, and its total antioxidant capacity as determined by two independent assays [1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)] was similar to that of the original garlic samples. Wistar rats (35) were randomly divided into five diet groups, named control, Chol, Garlic500, Garlic750, and Garlic1000. Control rats were fed basal diet (BD), which included wheat starch, casein, soybean oil, and vitamin and mineral mixtures. To the BD of the Chol group was added 1% of cholesterol. To the BD of the other three groups (Garlic500, Garlic750, and Garlic1000) were added 1% of cholesterol and commercial garlic equal to 500, 750, and 1000 mg of raw garlic per kilogram of animal weight. After 4 weeks of the experiment only in rats from the Garlic500 group were a significant hindering in the rise in plasma lipids and also a significant hindering in a decrease of plasma antioxidant activity registered. A significant decrease in plasma circulating fibrinogen and an increase in the clotting time were found in the same group of rats (P < 0.05 in both cases). The fibrinogenolytic effect of garlic diets was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the fibrinogen fraction of Garlic500 the 66, 24, and 14 kDa protein bands were detected with weaker protein intensity than in the corresponding ones in the Garlic750 and Garlic1000 diet groups. In conclusion, the positive influences of commercial garlic on plasma lipids, proteins, antioxidant activity, and some indices of blood coagulation are dose-dependent. Therefore, commercial garlic (Elena, Zelazków, Poland) could be a valuable component of atherosclerosis-preventing diets only in optimal doses.     

Evaluation of adverse effects of long-term orally administered carprofen in dogs

OBJECTIVE: To evaluate the adverse effects of carprofen in dogs after oral administration for 2 months. DESIGN: Prospective, randomized, blinded, placebo-controlled clinical trial. ANIMALS: 22 dogs with osteoarthritis in the hip or elbow joint. PROCEDURE: 13 dogs received orally administered carprofen daily for 2 months, and 9 dogs received a placebo for 2 months. Dogs were weighed, and serum and urine samples were collected before initiation of treatment and 4 and 8 weeks after initiation of treatment. Serum concentrations of total protein, albumin, urea, and creatinine and serum activities of alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were measured. Urinary ALP-to-creatinine, gamma-glutamyltransferase (GGT)-to-creatinine, and protein-to-creatinine ratios were calculated. Dogs were observed by owners for adverse effects. RESULTS: Serum protein and albumin concentrations were lower in treated dogs than in those that received placebo at 4 weeks, but not at 8 weeks. No changes were observed in serum urea or creatinine concentrations; ALP or ALT activity; or urinary ALP-to-creatinine, GGT-to-creatinine, or protein-to-creatinine ratios. Dogs' weights did not change. Severity of vomiting, diarrhea, and skin reactions did not differ between groups, but appetite was better in dogs receiving carprofen than in dogs in the placebo group. CONCLUSIONS AND CLINICAL RELEVANCE: It is possible that the transient decreases in serum protein and albumin concentrations in dogs that received carprofen were caused by altered mucosal permeability of the gastrointestinal tract because no indications of renal or hepatic toxicity were observed. Carprofen appeared to be well tolerated by dogs after 2 months of administration.