Sequence determination of oligosaccharides and regular polysaccharides using NMR spectroscopy and a novel Web-based version of the computer program CASPER.

A WWW-interface to a program for structure elucidation of oligo- and polysaccharides using NMR data, CASPER, is presented. The interface and the underlying program have been extensively tested using published data and it was able to simulate 13C NMR spectra of >200 structures with an average error of about 0.3 ppm/resonance. When applied to the repeating units of Escherichia coli O-antigens the published structures were found among the five highest ranked structures in 75% of the cases. The average deviation between calculated and experimental 13C chemical shifts was 0.45 ppm. Oligosaccharide spectra were calculated with even better accuracy (0.23 ppm/resonance) and the correct structure was ranked 1st or 2nd in all the cases examined. Additional NMR experiments that may be required to distinguish between candidate structures are aided by the assignments provided by the program. This computational approach is also suitable for use in structural confirmation of chemically or enzymatically synthesized oligosaccharides. The program is found at http://www.casper.organ.su.se/casper.

Modeling carbon turnover in five terrestrial ecosystems in the boreal zone using multiple criteria of acceptance.

Estimates of carbon fluxes and turnover in ecosystems are key elements in the understanding of climate change and in predicting the accumulation of trace elements in the biosphere. In this paper we present estimates of carbon fluxes and turnover times for five terrestrial ecosystems using a modeling approach. Multiple criteria of acceptance were used to parameterize the model, thus incorporating large amounts of multi-faceted empirical data in the simulations in a standardized manner. Mean turnover times of carbon were found to be rather similar between systems with a few exceptions, even though the size of both the pools and the fluxes varied substantially. Depending on the route of the carbon through the ecosystem, turnover times varied from less than one year to more than one hundred, which may be of importance when considering trace element transport and retention. The parameterization method was useful both in the estimation of unknown parameters, and to identify variability in carbon turnover in the selected ecosystems.

Modeller subjectivity and calibration impacts on hydrological model applications: an event-based comparison for a road-adjacent catchment in south-east Norway.

Identifying a 'best' performing hydrologic model in a practical sense is difficult due to the potential influences of modeller subjectivity on, for example, calibration procedure and parameter selection. This is especially true for model applications at the event scale where the prevailing catchment conditions can have a strong impact on apparent model performance and suitability. In this study, two lumped models (CoupModel and HBV) and two physically-based distributed models (LISEM and MIKE SHE) were applied to a small catchment upstream of a road in south-eastern Norway. All models were calibrated to a single event representing typical winter conditions in the region and then applied to various other winter events to investigate the potential impact of calibration period and methodology on model performance. Peak flow and event-based hydrographs were simulated differently by all models leading to differences in apparent model performance under this application. In this case-study, the lumped models appeared to be better suited for hydrological events that differed from the calibration event (i.e., events when runoff was generated from rain on non-frozen soils rather than from rain and snowmelt on frozen soil) while the more physical-based approaches appeared better suited during snowmelt and frozen soil conditions more consistent with the event-specific calibration. This was due to the combination of variations in subsurface conditions over the eight events considered, the subsequent ability of the models to represent the impact of the conditions (particularly when subsurface conditions varied greatly from the calibration event), and the different approaches adopted to calibrate the models. These results indicate that hydrologic models may not only need to be selected on a case-by-case basis but also have their performance evaluated on an application-by-application basis since how a model is applied can be equally important as inherent model structure.

Role of Helicobacter pylori rfaJ genes (HP0159 and HP1416) in lipopolysaccharide synthesis.

The genome of Helicobacter pylori 26695 has been sequenced and the lipopolysaccharide (LPS) O sidechain of this strain has been shown to express both Lewis x and Lewis y units. To determine the role of HP0159 and HP1416, genes recognized as rfaJ homologs and implicated in LPS synthesis, isogenic mutants of H. pylori 26695 were generated. The LPS of mutant 26695::HP0159Kan did not express either Lewis epitope as detected by immunoblotting, whereas the control strain and 26695::HP1416Kan produced both epitopes. Structural analysis of the LPS of the mutants showed that HP0159 encodes an alpha(1,2/3)-glucosyltransferase whereas HP1416 encodes an alpha(1,2/4)-glucosyltransferase.

Structure of the O-polysaccharide from the lipopolysaccharide from Vibrio cholerae O6.

The O-polysaccharide from Vibrio cholerae O6 was isolated from the LPS by mild-acid hydrolysis and has been investigated by sugar and methylation analysis and NMR spectroscopy. The polysaccharide was also depolymerized with aqueous hydrofluoric acid to give the repeating unit and multiples thereof. The O-polysaccharide had the following tetrasaccharide repeating unit. Two O-acetyl groups are present, one of them making the GlcNAc residue fully substituted and the steric crowding considerable at the branching residue.

Structure of a highly phosphorylated O-polysaccharide of Proteus mirabilis O41.

A highly phosphorylated O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Proteus mirabilis O41 followed by GPC. The initial and dephosphorylated polysaccharides and phosphorylated products from two sequential Smith degradations were studied by (1)H, (13)C and (31)P NMR spectroscopy and ESI-MS. The O-polysaccharide was found to have a tetrasaccharide repeating unit containing one ribitol phosphate (presumably d-Rib-ol-5-P) and two ethanolamine phosphate (Etn-P) groups, one of which is present in the stoichiometric amount and the other in a nonstoichiometric amount. The following structure of the O-polysaccharide was established:

Quantifying the hydrological impact of simulated changes in land use on peak discharge in a small catchment.

A physically-based, distributed hydrological model (MIKE SHE) was used to quantify overland runoff in response to four extreme rain events and four types of simulated land use measure in a catchment in Norway. The current land use in the catchment comprises arable lands, forest, urban areas and a stream that passes under a motorway at the catchment outlet. This model simulation study demonstrates how the composition and configuration of land use measures affect discharge at the catchment outlet differently in response to storms of different sizes. For example, clear-cutting on 30% of the catchment area produced a 60% increase in peak discharge and a 10% increase in total runoff resulting from a 50-year storm event in summer, but the effects on peak discharge were less pronounced during smaller storms. Reforestation of 60% of the catchment area was the most effective measure in reducing peak flows for smaller (2-, 5- and 10-year) storms. Introducing grassed waterways reduced water velocity in the stream and resulted in a 28% reduction in peak flow at the catchment outlet for the 50-year storm event. Overall, the results indicate that the specific effect of land use measures on catchment discharge depends on their spatial distribution and on the size and timing of storm events.

Structure of the O-polysaccharide of Vibriocholerae O43 containing a new monosaccharide derivative, 4-(N-acetyl-l-allothreonyl)amino-4,6-dideoxy-D-glucose.

The O-polysaccharide of Vibrio cholerae O43 was studied using chemical analyses, triflic acid solvolysis and 2D NMR spectroscopy, including (1)H/(1)H COSY, TOCSY, NOESY and (1)H/(13)C gradient-selected HSQC experiments. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: →3)-ß-D-Quip4NAcyl-(1→3)-α-D-GalpNAcA-(1→4)-α-D-GalpNAc-(1→3)-α-D-QuipNAc-(1→ where D-QuiNAc stands for 2-acetamido-2,6-dideoxy-D-glucose, D-Qui4NAcyl for 4-(N-acetyl-L-allothreonyl)amino-4,6-dideoxy-D-glucose and D-GalNAcA for 2-acetamido-2-deoxy-D-galacturonic acid.

Arctic vegetation damage by winter-generated coal mining pollution released upon thawing.

Acid mine drainage (known as AMD) is a well-known environmental problem resulting from the oxidation of sulfidic mine waste. In cold regions, AMD is often considered limited by low temperatures most of the year and observed environmental impact is related to pollution generated during the warm summer period. Here we show that heat generation within an oxidizing, sulfidic, coal-mining waste-rock pile in Svalbard (78 degrees N) is high enough to keep the pile warm (roughly 5 degrees C throughout the year) despite mean annual air temperatures below -5 degrees C. Consequently, weathering processes continue year-round within the waste-rock pile. During the winter, weathering products accumulate within the pile because of a frozen outer layer on the pile and are released as a flush within 2 weeks of soil thawing in the spring. Consequently, spring runoff water contains elevated concentrations of metals. Several of these metals are taken up and accumulated in plants where they reach phytotoxic levels, including aluminum and manganese. Laboratory experiments document that uptake of Al and Mn in native plant species is highly correlated with dissolved concentrations. Therefore, future remedial actions to control the adverse environmental impacts of cold region coal-mining need to pay more attention to winter processes including AMD generation and accumulation of weathering products.

The structure of the O-chain of the lipopolysaccharide of a prototypal diarrheagenic strain of Hafnia alvei that has characteristics of a new species under the genus Escherichia.

The structure of the O-polysaccharide of the lipopolysaccharide from a diarrheal strain isolated in Bangladesh was studied with sugar, and methylation analysis, NMR spectroscopy, mass spectrometry and partial acid hydrolysis. The strain was first designated as Hafnia alvei, but later found to be a possible new species in the genus Escherichia. Two different polysaccharides were detected, a major and a minor one. The structure of the major polysaccharide is given below, while the structure of the minor one was not investigated. The structure of the repeating unit was established as The structure does not resemble any of the previously investigated lipopolysaccharide O-chains from Escherichia coli or H. alvei, but could fit in either group based on types of sugar residues and acidity. Phenotypic microbiological studies cannot definitely assign it to either species of the two genera. Genetic hybridization studies indicate that the Bangladeshi isolates may require a new species designation under the genus Escherichia.

A unique variant of streptococcal group O-antigen (C-polysaccharide) that lacks phosphocholine.

Streptococcus mitis strain SK598, which represents a subgroup of biovar 1, possesses a unique variant of the C-polysaccharide found in the cell wall of all strains of Streptococcus pneumoniae and in some strains of S. mitis. This new variant lacks the choline methyl groups in contrast to the previously characterized forms of C-polysaccharide, which all contain one or two choline residues per repeat. The following structure of the repeating unit of the SK598 polysaccharide was established: where AAT is 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose. This structure is identical to the double choline-substituted form of C-polysaccharide, except that it is substituted with ethanolamine instead of choline. This extends the number of recognized C-polysaccharide variants to four.

Frequencies of lipopolysaccharide-defined epitopes in Haemophilus influenzae type b and non-typable isolates determined with monoclonal antibodies.

OBJECTIVE: To investigate the frequency of expression and stability of saccharide epitopes in 178 Haemophilus influenzae (39 type b and 138 non-typable) isolates from blood, cerebrospinal fluid, nasopharynx, pharynx, middle ear, conjunctiva, and pleural and bronchial fluid from symptomatic and asymptomatic children using five murine monoclonal antibodies (MAbs, MAHI 3, 4, 6, 8, 10) specific for the oligosaccharide moiety of the lipopolysaccharide (LPS) of H. influenzae, which recognize defined saccharide structures. METHODS: A whole bacteria enzyme immunoassay (EIA) and colony dot immunoblotting were used to determine the frequency of expression and stability of saccharide epitopes in the 178 H. influenzae isolates. RESULTS: Six main groups of strains were differentiated based on the EIA binding pattern with the MAbs: group A, reactive with all five MAbs (MAHI 3, 4, 6, 8 and 10); group B, reactive with four MAbs (MAHI 3, 6, 8 and 10); group C, reactive with three MAbs (MAHI 3, 6 and 8); group D, reactive with three MAbs (MAHI 3, 6 and 10); group E, reactive with two MAbs (MAHI 3 and 10); group F, reactive with MAb MAHI 3. Group B was the most common classification overall. None of the strains remained non-reactive. The frequencies of the binding patterns among the isolates obtained from different sources appeared to be statistically similar in most of the cases. Indications of phase variation of the LPS epitopes were observed with all the MAbs for strains obtained from all clinical sources as evaluated by colony dot immunoblotting. One of the epitopes displayed 22% phase variation, while four other epitopes were variably expressed, with about 50% on-off expression. CONCLUSIONS: This set of MAbs showed 100% reactivity among the isolates, in both EIA and colony dot immunoblotting, and allowed us to differentiate strains based on the LPS phenotype by whole bacteria EIA. Phase variation was indicated among all the isolates, independent of the source of isolation, and for all five MAbs. The LPS of isolates from different clinical sources often expressed some of the epitopes recognized by the MAbs, and most of the LPS phenotypes appeared at similar frequencies among isolates.

Phenotypic variation in molecular mimicry between Helicobacter pylori lipopolysaccharides and human gastric epithelial cell surface glycoforms. Acid-induced phase variation in Lewis(x) and Lewis(y) expression by H. Pylori lipopolysaccharides.

Helicobacter pylori is an important gastroduodenal pathogen of humans whose survival in the gastric environment below pH 4 is dependent on bacterial production of urease, whereas above pH 4 urease-independent mechanisms are involved in survival, but that remain to be elucidated fully. Previous structural investigations on the lipopolysaccharides (LPSs) of H. pylori have shown that the majority of these surface glycolipids express partially fucosylated, glucosylated, or galactosylated N-acetyllactosamine (LacNAc) O-polysaccharide chains containing Lewis(x) (Le(x)) and/or Lewis(y) (Le(y)), although some strains also express type 1 determinants, Lewis(a), Lewis(b), and H-1 antigen. In this study, we investigated acid-induced changes in the structure and composition of LPS and cellular lipids of the genome-sequenced strain, H. pylori 26695. When grown in liquid medium at pH 7, the O-chain consisted of a type 2 LacNAc polysaccharide, which was glycosylated with alpha-1-fucose at O-3 of the majority of N-acetylglucosamine residues forming Le(x) units, including chain termination by a Le(x) unit. However, growth in liquid medium at pH 5 resulted in production of a more complex O-chain whose backbone of type 2 LacNAc units was partially glycosylated with alpha L-fucose, thus forming Le(x), whereas the majority of the nonfucosylated N-acetylglucosamine residues were substituted at O-6 by alpha-D-galactose residues, and the chain was terminated by a Le(y) unit. In contrast, detailed chemical analysis of the core and lipid A components of LPS and analysis of cellular lipids did not show significant differences between H. pylori 26695 grown at pH 5 and 7. Although putative molecular mechanisms affecting Le(x) and Le(y) expression have been investigated previously, this is the first report identifying an environmental trigger inducing phase variation of Le(x) and Le(y) in H. pylori that can aid adaptation of the bacterium to its ecological niche.