Structural and hemostatic activities of a sulfated galactofucan from the brown alga Spatoglossum schroederi. An ideal antithrombotic agent?

The brown alga Spatoglossum schroederi contains three fractions of sulfated polysaccharides. One of them was purified by acetone fractionation, ion exchange, and molecular sieving chromatography. It has a molecular size of 21.5 kDa and contains fucose, xylose, galactose, and sulfate in a molar ratio of 1.0:0.5:2.0:2.0 and contains trace amounts of glucuronic acid. Chemical analyses, methylation studies, and NMR spectroscopy showed that the polysaccharide has a unique structure, composed of a central core formed mainly by 4-linked beta-galactose units, partially sulfated at the 3-O position. Approximately 25% of these units contain branches of oligosaccharides (mostly tetrasaccharides) composed of 3-sulfated, 4-linked alpha-fucose and one or two nonsulfated, 4-linked beta-xylose units at the reducing and nonreducing end, respectively. This sulfated galactofucan showed no anticoagulant activity on several "in vitro" assays. Nevertheless, it had a potent antithrombotic activity on an animal model of experimental venous thrombosis. This effect is time-dependent, reaching the maximum 8 h after its administration compared with the more transient action of heparin. The effect was not observed with the desulfated molecule. Furthermore, the sulfated galactofucan was 2-fold more potent than heparin in stimulating the synthesis of an antithrombotic heparan sulfate by endothelial cells. Again, this action was also abolished by desulfation of the polysaccharide. Because this sulfated galactofucan has no anticoagulant activity but strongly stimulates the synthesis of heparan sulfate by endothelial cells, we suggested that this last effect may be related to the "in vivo" antithrombotic activity of this polysaccharide. In this case the highly sulfated heparan sulfate produced by the endothelial cells is in fact the antithrombotic agent. Our results suggested that this sulfated galactofucan may have a potential application as an antithrombotic drug.     

Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-TOF MS: a contribution to understanding zona pellucida structure

The mammalian oocyte is encased by a transparent extracellular matrix, the zona pellucida (ZP), which consists of three glycoproteins, ZPA, ZPB, and ZPC. The glycan structures of the porcine ZP and the complete N-glycosylation pattern of the ZPB/ZPC oligomer has been recently described. Here we report the N-glycan pattern and N-glycosylation sites of the porcine ZP glycoprotein ZPA of an immature oocyte population as determined by a mass spectrometric approach. In-gel deglycosylation of the electrophoretically separated ZPA protein and comparison of the pattern obtained from the native, the desialylated and the endo-beta-galactosidase-treated glycoprotein allowed the assignment of the glycan structures by MALDI-TOF MS by considering the reported oligosaccharide structures. The major N-glycans are neutral biantennary complex structures containing one or two terminal galactose residues. Complex N-glycans carrying N-acetyllactosamine repeats are minor components and are mostly sialylated. A significant signal corresponding to a high-mannose type chain appeared in the three glycan maps. MS/MS analysis confirmed its identity as a pentamannosyl N-glycan. By the combination of tryptic digestion of the endo-beta-galactosidase-treated ZP glycoprotein mixture and in-gel digestion of ZPA with lectin affinity chromatography and reverse-phase HPLC, five of six N-glycosylation sites at Asn(84/93), Asn268, Asn316, Asn323, and Asn530 were identified by MS. Only one site was found to be glycosylated in the N-terminal tryptic glycopeptide with Asn(84/93.) N-glycosidase F treatment of the isolated glycopeptides and MS analysis resulted in the identification of the corresponding deglycosylated peptides.     

Point mutations abolishing the mannose-binding capability of boar spermadhesin AQN-1

The mannose-binding capability of recombinant wild-type boar spermadhesin AQN-1 and of its site-directed mutants in the highly-conserved region around of the single glycosylation site (asparagine 50) of some spermadhesins, where the carbohydrate binding site has been proposed to be located, was checked using a solid-phase assay and a biotinylated mannose ligand. Substitution of glycine 54 by amino acids bearing an unipolar side chain did not cause significant decrease in the mannose-binding activity. However, amino acids with uncharged polar side chains or having a charged polar side chain abolished the binding of biotinylated mannose to the corresponding AQN-1 mutants. The results suggest that the higher surface accessibility of amino acids possessing polar side chains compared to those bearing nonpolar groups may sterically interfere with monosaccharide binding. The location of the mannose-binding site in AQN-1 appears to be topologically conserved in other heparin-binding boar spermadhesins, i.e., AQN-3 and AWN, but departs from the location of the mannose-6-phosphate-recognition site of PSP-II. This indicates that different spermadhesin molecules have evolved non-equivalent carbohydrate-binding capabilities, which may underlie their distinct patterns of biological activities.     

The highly conserved LepA is a ribosomal elongation factor that back-translocates the ribosome

The ribosomal elongation cycle describes a series of reactions prolonging the nascent polypeptide chain by one amino acid and driven by two universal elongation factors termed EF-Tu and EF-G in bacteria. Here we demonstrate that the extremely conserved LepA protein, present in all bacteria and mitochondria, is a third elongation factor required for accurate and efficient protein synthesis. LepA has the unique function of back-translocating posttranslocational ribosomes, and the results suggest that it recognizes ribosomes after a defective translocation reaction and induces a back-translocation, thus giving EF-G a second chance to translocate the tRNAs correctly. We suggest renaming LepA as elongation factor 4 (EF4).     

Quantification of campylobacter species cross-contamination during handling of contaminated fresh chicken parts in kitchens

Numerous outbreak investigations and case-control studies for campylobacteriosis have provided evidence that handling Campylobacter-contaminated chicken products is a risk factor for infection and illness. There is currently extremely limited quantitative data on the levels of Campylobacter cross-contamination in the kitchen, hindering risk assessments for the pathogen commodity combination of Campylobacter and chicken meat. An exposure assessment needs to quantify the transfer of the bacteria from chicken to hands and the kitchen environment and from there onto ready-to-eat foods. We simulated some typical situations in kitchens and quantified the Campylobacter transfer from naturally contaminated chicken parts most commonly used in Germany. One scenario simulated the seasoning of five chicken legs and the reuse of the same plate for cooked meat. In another, five chicken breast filets were cut into small slices on a wooden board where, without intermediate cleaning, a cucumber was sliced. We also investigated the transfer of the pathogen from chicken via hands to a bread roll. The numbers of Campylobacter present on the surfaces of the chicken parts, hands, utensils, and ready-to-eat foods were detected by using Preston enrichment and colony counting after surface plating on Karmali agar. The mean transfer rates from legs and filets to hands were 2.9 and 3.8%. The transfer from legs to the plate (0.3%) was significantly smaller (P < 0.01) than the percentage transferred from filets to the cutting board and knife (1.1%). Average transfer rates from hands or kitchen utensils to ready-to-eat foods ranged from 2.9 to 27.5%.     

Optimal offspring size in a small mammal: an exception to the tradeoff invariant life-history rule

Offspring size and number were examined in a captive population of wild guinea pigs ( Cavia aperea ), and findings were compared with models of optimal offspring size for small litters. Median and modal litter size was two, regardless of maternal size or parity. Females producing their second litter tended to have litters that were larger than average. In contrast, young females that were still growing never had litters that were larger than average. Mean offspring size decreased and variation in offspring size tended to decrease with increasing litter size. Optimal offspring size models, in which offspring survival depended on the amount of resources invested, as well as litter size, predict such a trend. Little support was found for Charnov and Downhower's (1995) tradeoff invariant life-history rule that the range in offspring sizes between litters is inversely proportional to the size of the litter. Cavia aperea may be an exception to this rule because pup mass at birth did not reflect total reproductive investment, because conversion of resources into litter mass may not be linearly related to litter size and because resources were not equally partitioned among offspring within large litters. Experimental data are needed to determine the relevance of these results among mammals in general.     

Transgenic papaya: a useful platform for oral vaccines

Planta - Transgenic papaya callus lines expressing the components of the S3Pvac vaccine constitute a stable platform to produce an oral vaccine against cysticercosis caused by Taenia solium or T....     

Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in the pig.

Competitive inhibition of sperm to explants of the oviductal epithelium was used to study the complementary receptor system that may be involved in the establishment of the oviductal sperm reservoir in the pig. Sperm binding to the oviductal explants is expressed as Binding Index (BI = sperm cells/0.01 mm(2)). From a set of glycoproteins with known oligosaccharide structures, only asialofetuin and ovalbumin showed inhibitory activity, indicating that ovalbumin may block high affinity binding sites (IC(50) congruent with 1.3 microM) and asialofetuin low affinity sites (IC(50) congruent with 18 microM) of the complementary receptor systems, whereas fetuin carrying terminal sialic acid has no effect. Ovalbumin glycopeptides were isolated by Con A affinity chromatography and reverse-phase HPLC following tryptic digestion. Glycopeptides and enzymatically released glycans were analyzed by MS, and were shown to represent preferentially the two high mannose type glycans (Man)(5)(GlcNAc)(2) and (Man)(6)(GlcNAc)(2), and as a minor component the hybrid type glycan (Hex)(4)(GlcNAc)(5). Glycopeptides (84% inhibition) and glycans (81% inhibition) significantly reduced sperm-oviduct binding at a concentration of 3 microM, whereas the deglycosylated peptides showed no inhibitory activity. Mannopentaose (IC(50) congruent with 0.8 microM) representing the oligomannose residue of the high mannose glycans of ovalbumin was as effective as ovalbumin. These data indicate that the carbohydrate-based mechanisms underlying the formation of the oviductal sperm reservoir in the pig is the result of the concerted action of at least the high-affinity binding sites for oligomannose or nonreducing terminal mannose residues and low-affinity binding of galactose.