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Globally, boreal forests act as important carbon sinks, however, drought and forest management could substantially alter the sink strength, though the controlling mechanisms of drought and management remain unclear. In this study, we combined the detailed process-based CoupModel with multiple measurements to study the impacts of recent drought and forest thinning on a boreal forest during 2018-2021. CoupModel after calibration showed high ability to represent the dynamics of long-term net ecosystem exchange and its responses to environmental changes. The model simulation showed that the canopy temperature exacerbated the dominant role in regulating the boreal forest growth during the 2018 extreme drought year with slight increase in the annual mean net carbon uptake by 76.65 g C/m 2 /yr compared to 2017. The posterior model simulations ensemble suggested that thinning of trees in 2019-2020 caused the boreal forest in 2020 to be a sink to slight source ([-229.95, 94.90] g C/m 2 /yr, 90 % confidence interval), while the observations depicted a small source (69.35 g C/m 2 /yr). Moreover, rapid recovery of the boreal forest to a carbon sink was found in 2021, though remaining smaller than the carbon sink in 2017. Overall, the negative impacts from drought and harvest (2018-2021) were found to have offset the positive impacts from climate by 8 % - 92 %, on the net carbon uptake. This study highlights the resilience of boreal forests as carbon sink and provides new insights into the boreal forests' responses to both climate change and management., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (© 2013.)

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)-β-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., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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.

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:

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.

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.

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.

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.

The exocellular polysaccharide S-7, a heteropolysaccharide from Azotobacter indicus var. myxogenes has been studied using methylation analysis, Smith degradation, partial acid hydrolysis, NMR spectroscopy and mass spectrometry as the principal methods. It is concluded that the repeating unit has the following structure: [structure: see text] The absolute configuration of the deoxyhexuronic acid was deduced from 1H NMR chemical shifts and is most likely D. Approximately two O-acetyl groups per repeating unit are present, one of which is presumably on the Rha residue. The structure bears great resemblance to another polysaccharide, recently studied, produced by Sphingomonas paucimobilis I-886.

A lipopolysaccharide (LPS) was isolated by hot phenol-water extraction from Helicobacter pylori strain D4 and found to contain no fucosylated poly-N-acetyllactosamine chain typical of most H. pylori strains studied but a homopolymer of D-glycero-D-manno-heptose (DD-Hep). The heptan attached to a core oligosaccharide was released by mild acid degradation of the LPS, and the following structure of the trisaccharide-repeating unit was established by chemical methods and 1H and 13C NMR spectroscopy: --> 2)-D-alpha-D-Hepp-(1 --> 3)-D-alpha-D-Hepp-(1 --> 3)-D-alpha-D-Hepp-(1 -->. 1H NMR spectroscopy performed on small amounts of the intact LPS revealed the presence of the same polysaccharide in LPS of H. pylori strains D2 and D5, but not strain D10.

The O-specific polysaccharide (OPS) of Vibrio cholerae 08 was isolated by mild acid degradation of the lipopolysaccharide and studied by two-dimensional NMR spectroscopy, including NOESY and heteronuclear multiple-bond correlation (HMBC) experiments. The OPS was found to have a tetrasaccharide repeating unit with the following structure: --> 4)-beta-D-Glcp NAc3NAcylAN-(1 --> 4)-beta-D-Manp NAc3NAcAN-(1 --> 4)-alpha-L-Gulp NAc3NAcA-(1 --> 3) -beta-D-QuipNAc4NAc-(1 --> where QuiNAc4NAc is 2,4-diacetamido-2,4,6-trideoxyglucose, GlcNAc3NAcylAN is 2-acetamido-3-(N-formyl-L-alanyl)amino-2,3-dideoxyglucuronamide, ManNAc3NAcAN is 2,3-diacetamido-2,3-dideoxymannuronamide, and GulNAc3NAcA is 2,3-diacetamido-2,3-dideoxyguluronic acid. The OPS was stable towards acid hydrolysis and solvolysis with anhydrous hydrogen fluoride, but could be cleaved selectively with trifluoromethanesulfonic (triflic) acid by the glycosidic linkages of beta-QuiNAc4NAc and alpha-GulNAc3NAcA. The structures of the oligosaccharides obtained that were elucidated by electrospray ionization (ESI) MS and NMR spectroscopy, confirmed the OPS structure.

The cell wall of Streptococcus mitis biovar 1 strain SK137 contains the C-polysaccharide known as the common antigen of a closely related species Streptococcus pneumoniae, and a teichoic acid-like polysaccharide with a unique structure. The two polysaccharides are different entities and could be partially separated by gel chromatography. The structures of the two polysaccharides were determined by chemical methods and by NMR spectroscopy. The teichoic acid-like polymer has a heptasaccharide phosphate repeating unit with the following structure: The structure neither contains ribitol nor glycerol phosphate as classical teichoic acids do, thus we have used the expression teichoic acid-like for this polysaccharide. The following structure of the C-polysaccharide repeating unit was established: where AAT is 2-acetamido-4-amino-2,4, 6-trideoxy-D-galactose. It has a carbohydrate backbone identical to that of one of the two structures of C-polysaccharide previously identified in S. pneumoniae. C-polysaccharide of S. mitis is characterized by the presence, in each repeating unit, of two residues of phosphocholine and both galactosamine residues in the N-acetylated form. Immunochemical analysis showed that C-polysaccharide constitutes the Lancefield group O antigen. Studies using mAbs directed against the backbone and against the phosphocholine moiety of the C-polysaccharide revealed several different patterns of these epitopes among 95 S. mitis and Streptococcus oralis strains tested and the exclusive presence of the group O antigen in the majority of S. mitis biovar 1 strains.

The acidic polysaccharide from Serratia marcescens serogroup O14:K12 was analyzed by means of chemical studies and NMR spectroscopy and its repeating unit structure found to be carbohydrate sequence [see text] O-Acetyl groups are proposed to be present in non-stoichiometric amounts on O-6 on one of the hexose residues in the main chain.

Lipopolysaccharides (LPS) were isolated by hot phenol-water extraction from Danish Helicobacter pylori strains D1, D3, and D6, which were nontypeable using a variety of anti-Lewis and anti-blood-group monoclonal antibodies. An atypical O-chain polysaccharide (PS) was liberated from the LPS of the three strains by acid under mild conditions and found to contain D-rhamnose (D-Rha), L-rhamnose (L-Rha), and a branched sugar, 3-C-methyl-D-mannose (D-Man3CMe). The last sugar, which has not hitherto been found in Nature, was identified using GLC-MS of the derived alditol acetate and the partially methylated alditol acetate, and (1)H and (13)C NMR spectroscopy, including NOESY and (1)H,(13)C HMBC experiments. The following structure of the trisaccharide repeating unit of the PS was established: -->2)-alpha-D-Manp3CMe-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-D- Rhap-(1-- >. In contrast to the pathogenic importance of the Lewis antigen mimicry exhibited by the PS of H. pylori strains previously investigated, the biological relevance of the atypical PS for H. pylori pathogenesis is unclear. The production of a differing surface PS may represent a form of antigenic variation by these particular H. pylori strains and/or may reflect the adaptation of these strains to a particular human population.

The structure of the pneumococcal common antigen, C-polysaccharide, from a noncapsulated pneumococcal strain, CSR SCS2, was studied using 1H-NMR, 13C-NMR and 31P-NMR spectroscopy. The dependence of NMR chemical shifts on the variation in pD was also studied. It was established that the C-polysaccharide is composed of a backbone of tetrasaccharide-ribitol repeating units that are linked to each other by a phosphodiester linkage between position 5 of a D-ribitol residue and position 6 of a beta-D-glucopyranosyl residue. The polysaccharide is substituted with one residue of phosphocholine at position 6 of the 4-substituted 2-acetamido-2-deoxy-alpha-D-galactopyranosyl residue. Both galactosamine residues in the polysaccharide are N-acetylated. O)-P-Cho | 6 6)-beta-D-Glcp-(1-->3)-alpha-AATp-(1-->4)-alpha-D-GalpNAc-(1-->3)- bet a-D-GalpNAc-(1-->1)-D-ribitol-5-P-(O--> where AAT is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose and Cho is choline. This structure differs, concerning phosphocholine substituents and N-acetylation, from those reported previously for pneumococcal C-polysaccharide [Jennings, H.J., Lugowski, C. & Young, N.M. (1980) Biochemistry 19, 4712-4719; Fischer, W., Behr, T., Hartmann, R., Peter-Katalinic, J. & Egge, H. (1993) Eur. J. Biochem. 215, 851-857; Kulakowska, M., Brisson, J.-R., Griffith, D.W., Young, N.M. & Jennings, H.J. (1993) Can. J. Chem. 71, 644-648]. The structures of the C-polysaccharides present in three pneumococcal types were also examined. They contain one (in 18B) or two (in 32F and 32A) phosphocholine residues in the repeating unit. The degree of substitution was not determined. The backbone of all examined C-polysaccharides was identical and in all cases both galactosamine residues appeared to be N-acetylated.

In contrast to other Helicobacter pylori strains, which have serologically detectable Lewis(x)+ (Le(x)) and Lewis(y)++ (++Le(y)) antigenic determinants in the O-specific polysaccharide chains of the lipopolysaccharides, H. pylori AF1 and 007 were non-typable with anti-Le(x) and anti-Le(y) antibodies. The carbohydrate portions of the lipopolysaccharides were liberated by mild acid hydrolysis and subsequently studied by sugar and methylation analyses, 1H-NMR spectroscopy and electrospray ionization-mass spectrometry. Compared with each other, and with lipopolysaccharides of strains studied previously, the lipopolysaccharides of both AF1 and 007 showed similarities, but also differences, in the structures of the core region and O-specific polysaccharide chains. The O-specific polysaccharide chains of both strains consisted of a short or long polyfucosylated poly-N-acetyl-beta-lactosamine chains, which were distinguished from those of other strains by a high degree of fucosylation producing a polymeric Le(x)chain terminating with Le(x) or Le(y) units:[sequence: see text] where n = 0 or 1 in strain AF1 and 0 in strain 007, m = 0-2, 6-7 in strain AF1 and m = 0-2, 6-7 or approximately 40 in strain 007, the medium-size species being predominant. Therefore, compared with other strains, the lack of reactivity of lipopolysaccharide of H. pylori AF1 and 007 with anti-Le(x) and anti-Le(y) may reflect the presence of a polymeric Le(x) chain and has important implications for serological and pathogenesis studies. As the substitution pattern of a D-glycero-D-manno-heptose residue in the outer core varied in the two strains, and an extended DD-heptan chain was present in some lipopolysaccharide species but not in others, this region was less conservative than the inner core region. The inner core L-glycero-D-manno-heptose region of both strains carried a 2-aminoethyl phosphate group, rather than a phosphate group, as reported previously for other H. pylori strains.

Moraxella catarrhalis has recently been shown to be both widespread and pathogenic, in contrast to previous reports. Several factors have been suggested as virulence factors, lipopolysaccharide (LPS) being one. Recent studies have shown the LPS to be without the O-chain, i.e. the polysaccharide part, and to have specific structural features corresponding to each of the three serogroups, A, B and C. The structures resemble in many respects those present in other Gram-negative nonenteric bacteria, with a galabiosyl element as a prominent common denominator. The presence of such common structures suggests that the LPS of these bacteria might be a part of a mechanism of survival for bacteria colonizing the human host.

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.

The structure of a short-chain lipopolysaccharide (LPS) of Vibrio cholerae O22 strain 169-68, that cross-reacts with V. cholerae O139 Bengal, was elucidated. The structure differs in detail from that reported on another strain of O22 [A.D. Cox, J-R. Brisson, P. Thibault and M.B. Perry, Carbohydr. Res., 304 (1997) 191-208]. The similarity and difference between the LPS structures of the two strains as well as between O22 and O139 are discussed.

The structures of the capsular polysaccharides from Streptococcus pneumoniae types 32F and 32A have been determined by means of NMR spectroscopy as the principal method. It is concluded that both polysaccharides are composed of tetrasaccharide repeating units with a phosphorylcholine (PCho) group linked to the 3-position of the 4-substituted beta-L-rhamnose (Rha) residue. Both polysaccharides are substituted with one O-acetyl group at the 2-position of the same beta-L-rhamnose residue. In addition, the type-32A polysaccharide is substituted with another O-acetyl group at the 4-position of the 2,3-disubstituted alpha-D-glucose residue, i.e. the branch-point residue. An unusual detail in the structure is that the side chain is composed of a rhamnosyl phosphate. [chemical structure: see text] In the type-32F polysaccharide R=H, and in the type-32A polysaccharide R=Ac. The structure of C-polysaccharide found in our preparations of type-32F and type-32A capsular polysaccharides is in agreement with that published previously for the pneumococcal common antigen C-polysaccharide [Fischer, W., Behr, T., Hartmann, R., Peter-Katalinic, J. & Egge, H. (1993) Eur. J. Biochem. 215, 851-857; Kulakowska, M., Brisson, J.-R., Griffith, D. W., Young, N. M. & Jennings, H. J. (1993) Can. J. Chem. 71, 644-648].

The O-specific polysaccharide of Vibrio cholerae 0155 was studied by sugar and methylation analyses, dephosphorylation with 48% hydrofluoric acid, 1H- and 13C-NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, and heteronuclear single-quantum coherence (HSQC) experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established: carbohydrate sequence [see text]. An unusual component, D-galactose 4,6-cyclophosphate, has been reported previously as a component of the capsular polysaccharide and O-antigen of V. cholerae O139 Bengal and appears to be responsible for the known serological cross-reactivity between V. cholerae O139 and O155.

The CASPER program which is used for determination of the structure of oligo- and polysaccharides has been extended. It can now handle a reduced number of experimental signals from an NMR spectrum in the comparison to the simulated spectra of structures that it generates, an improvement which is of practical importance since all signals in NMR spectra cannot always be identified. Furthermore, the program has been enhanced to simulate NMR spectra of multibranched oligo- and polysaccharides. The new developments were tested on four saccharides of known structure but of different complexity and were shown to predict the correct structures.

The structure of the capsular polysaccharide from Streptococcus pneumoniae type 18B has been determined using NMR spectroscopy and methylation analysis as the principal methods. It is concluded that the polysaccharide is composed of pentasaccharide repeating units with a glycerol phosphate substituting the 3-position of the branch point residue. The carbohydrate backbone in type 18B is identical to that in S. pneumoniae type 18F but without the O-acetyl groups present in that type. [formula: see text] In this structure, the absolute configuration of the glycerol phosphate moiety has not been determined but should be D, in analogy with that determined for the capsular polysaccharide from S. pneumoniae type 18A [T. Rundlöf, G. Widmalm, Anal. Biochem., 243 (1996) 228-233].

The structure of the capsular polysaccharide from Klebsiella type 52 has been elucidated using an improved and extended version of the computerised approach CASPER and NMR spectroscopy as principal methods. A previous suggestion to the structure but without the anomeric prefixes, could be shown correct [H. Björndal et al., Carbohydr. Res., 31 (1973) 93-100]. The polysaccharide has a hexasaccharide repeat with the following structure: [formula: see text]

A Vibrio cholerae O139 strain, MO10-T4, lacking capsular polysaccharide, produces a short-chain lipopolysaccharide (LPS), similar to enterobacterial SR strains. It was studied by acidic and alkaline degradation, dephosphorylation, sugar and methylation analysis, high-performance anion-exchange chromatography, one- and two-dimensional 1H-, 13C-, and 31P-NMR spectroscopy, and electrospray ionization mass spectrometry. The following structure was proposed for the core region of the LPS: [structure: see text] where PEtn stands for 2-aminoethyl phosphate, Fru for fructose, Hep for L-glycero-D-manno-heptose, and Kdo for 3-deoxy-D-manno-octulosonic acid; unless otherwise stated, the monosaccharide residues are D and present in the pyranose form. An O-acetyl group is present on a secondary position, tentatively O4 of the alpha-linked glucosyl group. Some LPS species contain an additional putative fructose residue whose location remains unknown. An O139-negative mutant strain, Bengal-2R, derived from V. cholerae O139, has also been investigated and shown to produce an O-antigen-lacking LPS similar to those from enterobacterial R strains, some of the LPS species containing the same core region as the strain MO10-T4 LPS and the other lacking the lateral heptose residue. The carbohydrate backbone core structure is the same for the V. cholerae O139 and V. cholerae O1 LPS, thus confirming the close relation between these bacteria; however, the 2-aminoethyl phosphate, the O-acetyl group, and the second fructose residue have not been reported for the O1 LPS. In the V. cholerae O139 strain MO10-T4 LPS, a short O-side chain is attached at position 3 of the 7-substituted heptose residue and has the same structure as one repeating unit of the V. cholerae O139 capsular polysaccharide. Some details of the structure proposed are at variance with those recently published for another V. cholerae O139 strain [Cox, A. D., Brisson, J.-R., Varma, V. & Perry, M. B. (1996) Carbohydr. Res. 290, 43-58; Cox, A. D. & Perry, M. B. (1996) Carbohydr. Res. 290, 59-65.]

The carbohydrate backbone of the Hafnia alvei strain Y166/91 lipopolysaccharide (LPS) was isolated by mild acid hydrolysis followed by gel permeation chromatography and studied by NMR spectroscopy and methylation analysis. Treatment with periodate and hypoiodite gave a modified polysaccharide which was also characterised. It was concluded that the polysaccharide has the following structure with two distinct regions, which are connected. The chain length parameters m and n were not determined but the ratio m/n is approximately 2. [-->3)-beta-D-Galp-1(1-->3)-alpha-D-Galp-(1-->]m[-->3)-alpha-D-Galp-(1-- >3)- beta-D-Galf-(1-->]n From the present data it is not possible to determine whether it is the Galp-Galp chain or the Galp-Galf chain that is connected to the core. The structure found here is identical to that suggested for the O-specific polysaccharide chain of Klebsiella pneumoniae O1K2 (NCTC 5055) LPS [O. Kol, J.-M. Wieruszeski, G. Strecker, J. Montreuil, and B. Fournet, Carbohydr. Res., 217 (1991) 117-125; O. Kol, J.-M. Wieruszeski, G. Strecker, B. Fournet, R. Zalisz, and P. Smets, Carbohydr. Res., 236 (1992) 339-344].

The absolute configuration of a sugar can be determined by gas-liquid chromatography of the acetylated or trimethylsilylated dithioacetals from 1-phenylethanethiol. The isolation of both enantiomers of 1-phenylethanethiol is also described. Using the acetates and both thiol reagents the absolute configuration of C-2 can be determined, provided it is a hydroxyl group, with great certainty. A new way of determining the absolute configuration of sugars, without references, is thereby provided. The sugars analysed include aldoses, deoxyaldoses, 2-acetamido-2-deoxyaldoses and uronic acids. The analysis is made using columns with non-chiral stationary phase and the electron impact mass spectra of the acetylated and trimethylsilylated bis(1-phenylethyl)dithioacetals are described.

The structure of the oligosaccharide part of the Haemophilus influenzae RM.118-28 lipopolysaccharide (LPS) has been investigated. The oligosaccharide was obtained from the LPS by mild acid hydrolysis followed by gel-permeation chromatography, and was studied by methylation analysis, NMR spectroscopy and mass spectrometry. The structure of the major compound, which is a hexasaccharide, is proposed as follows. [formula: see text] In the structure, Kdo is 3-deoxy-D-manno-octulosonic acid, PEtn is phosphoethanolamine, PCho is phosphocholine and L,D-Hep is L-glycero-D-manno-heptose. Electrospray-ionization mass spectrometry on O-deacylated LPS obtained after treatment with anhydrous hydrazine gave evidence for the presence of two minor compounds, which show additional substitution of the main structure with phosphate and PEtn, respectively. These substitutions have not been localized.

The oligosaccharides from the lipopolysaccharides of Moraxella catarrhalis serotype B, strain CCUG 3292, were isolated after mild acid hydrolysis and separated by high-performance anion-exchange chromatography. The structures of the oligosaccharides were established by fast atom bombardment mass spectrometry and nuclear magnetic resonance spectroscopy. It is concluded that the oligosaccharides comprise a mixture of mainly a nona- and a deca-saccharide. [formula: see text] Smaller amounts of undeca-saccharides and of truncated forms, namely, hexa-, hepta-, and octa-saccharides, were also detected.

The halophilic Archaeon Haloferax mediterranei exudes into the growth medium a high molecular weight sulfated polysaccharide. The structure of the repeating unit of this polymer was determined by a combination of glycose, methylation, and sulfate analysis, periodate oxidation, and 1D and 2D NMR spectroscopic analysis of the native and periodate-oxidised/reduced polysaccharides. The location of the sulfate group was established from the 1H and 13C NMR data. The structure of the repeating unit of the polysaccharide may be written as [formula: see text]

Mouse monoclonal antibodies MAHI 4 and MAHI 10 reactive with Haemophilus influenzae lipopolysaccharide (LPS), were generated by fusing mouse myeloma cells with spleen cells of mice immunized with H. influenzae strain RM.7004-XP-1. The antibody MAHI 4 reacted in whole-cell enzyme immunoassay (EIA) and colony-dot-immunoblotting with 20 of 123 H. influenzae strains and to a few other human Haemophilus spp. and Neisseria spp., but not to any Bordetella pertussis, B. parapertussis, Aeromonas spp. or Moraxella catarrhalis strains tested. This suggests a specific epitope accessible to recognition in just a few strains. This conclusion was supported by the data on binding of MAHI 4 to only three of 18 H. influenzae LPSs tested, but not to any Haemophilus ducreyi or enterobacterial LPSs. The antibody MAHI 10 bound to 80 of 123 strains of H. influenzae and to a few strains of Neisseria spp. and M. catarrhalis as evaluated by EIA and colony-dot-immunoblotting, which suggests an epitope accessible to recognition in 65% of the H. influenzae strains tested. The antibody MAHI 10 reacted with 10 of 18 H. influenzae LPSs as determined by EIA. By using polysaccharides, obtained after both mild acidic hydrolysis, strong alkali treatment, and dephosphorylation, as inhibitors of the antibodies binding to H. influenzae LPS antigens it was shown that phosphate groups were essential for the binding of MAHI 10 to LPS but they did not affect antigenic recognition by MAHI 4. None of the monoclonal antibodies bound to isolated lipid A, but the aggregation caused by the fatty acids of lipid A was essential for optimum epitope recognition. Enzymatic treatment of homologous LPSs with galactose-oxidase led to products which were between 20 to 30 times less effective as inhibitors of the binding of the MAHI 4 than the native LPSs. Taken together the results indicate that MAHI 4 has the following pentasaccharide as the epitope Gal beta 1-->2 Hep alpha 1-->2Hep alpha 1-->3Hep alpha 1--> Kdo(P). These results emphasize the importance of the terminal beta-Gal residue in the definition of the MAHI 4 specificity, and of the terminal phosphorylated saccharide residues of some of the Haemophilus LPSs for the MAHI 10 specificity.

Oxidative decarboxylation of peracetylated welan gum (S-130) with lead tetraacetate resulted in selective cleavage of the glucuronosidic linkages. Products of the degradation were reduced with sodium borohydride, O-deacetylated, and fractionated. Polymeric and oligomeric fractions were separated and analysed by 1H NMR spectroscopy and fast atom bombardment mass spectrometry, and were found to be monomers, dimers, and trimers of the repeating unit. Results show that this method may be used to liberate alditol-terminated multiples of the repeating unit of peracetylated glycuronans by cleavage and degradation of the uronic acid residues. The reaction sequence also confirms the recent finding that welan gum contains repeating units with randomly distributed terminal groups.

The structure of the O-antigenic polysaccharide from a strain of Aeromonas caviae, isolated from the stools of a patient with diarrhoea, has been investigated. Sugar analysis, methylation analyses, and a uronic acid degradation together with NMR spectroscopy were the principal methods used. The sequence of the sugar residues could be determined by NOESY and HMBC experiments. It is concluded that the polysaccharide is composed of pentasaccharide repeating units with the following structure: [sequence: see text]

The oligosaccharide part of the Vibrio salmonicida (strain NCMB 2262) lipopolysaccharide was isolated by mild acid hydrolysis followed by gel-permeation chromatography. The structure was established mainly by methylation analysis, mass spectrometry, and NMR spectroscopy. It is concluded that the oligosaccharide has the following structure, in which L-alpha-D-Hep p is L-glycero-alpha-D-manno-heptopyranose, D-alpha-D-Hepp is D-glycero-alpha-D-manno-heptopyranose, alpha-D-Fuc p4N is 4-amino-4,6-dideoxy-alpha-D-galactopyranose, alpha-NonA is 5-acetamidino-7-acetamido-3,5,7, 9-tetradeoxy-L-glycero-alpha-D-galacto-nonulosonic acid, BA is (R)-3-hydroxybutanoyl, and PEA is phosphoethanolamine. The substitution pattern of the branching heptosyl residue was deduced from 1H NMR chemical shifts and conformations of the branching region, obtained by molecular modelling. The absolute configuration for NonA was determined by NMR spectroscopy from NOE correlations to the neighbouring sugar and 13C NMR chemical shift data. It could also be shown that assignments of nonulosonic acids with the D-glycero-L-galacto configuration, reported by previous investigators, are erroneous and should be changed to L-glycero-D-galacto. The oligosaccharide is assumed to be linked to the 5-position of a Kdo residue, phosphorylated in the 4-position as observed for other lipopolysaccharides from Vibrionaceae. [formula: see text]

The O-specific polysaccharide of an Aeromonas trota strain was isolated by hydrolysis of the lipopolysaccharide at pH 4.5 followed by gel-permeation chromatography and found to consist of hexasaccharide repeating units containing D-galactose, L-rhamnose, 3,6-dideoxy-L-xylo-hexose (colitose, Col), 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-galactose in the ratios 1:1:2:1:1. Partial hydrolysis of the polysaccharide with 48% hydrofluoric acid resulted in selective removal of colitose to give a modified polysaccharide containing the other four sugar constituents. On the basis of methylation analysis and NMR spectroscopic studies of the initial and modified, colitose-free polysaccharide, it was concluded that the repeating unit of the O-specific polysaccharide has the following structure [sequence: see text] The known cross-reactivity between the strain studied and Vibrio cholerae O139 Bengal is substantiated by the presence of a common colitose-containing epitope shared by the O-specific polysaccharide of A. trota and the capsular polysaccharide of V. cholerae, which is thought to carry determinants of O-specificity.

The exocellular polysaccharide from Sphingomonas paucimobilis strain I-886 has been studied using methylation analysis, Smith degradation, partial acid hydrolysis, NMR spectroscopy, and mass spectrometry as the principal methods. It is concluded that the repeating unit has the following structure: [formula: see text] The absolute configuration of the uronic acid was deduced from 1H NMR chemical shifts and is most likely D. Some preparations of the polysaccharide also contain phosphate and O-acyl groups which have not been identified or localised.

A strain of Klebsiella pneumoniae was found as the only isolate with pathogenic potential from the stool of a two-year old patient with diarrhoea. A strong serological cross-reactivity with Shigella flexneri serotype 6 was demonstrated. The cross-reacting antigens were shown to reside in the cell wall lipopoly-saccharide. Studies of the pathogenic potential of the Klebsiella strain showed low level of invasion of HEp-2 cells. However, tests for adherence to HEp-2 cells as well as tests for toxin production were negative. The strain had several small plasmids and was multidrug resistant. These data do not form a sufficient basis for estimating the pathogenic potential of the organism. No K antigen was detected. The structure of the O-antigenic polysaccharide from the K. pneumoniae strain was investigated using methylation analysis, NMR spectroscopy, and Smith degradation as the principal methods. The O-antigenic polysaccharide has the following pentasaccharide repeating unit: -->3)- alpha -L-Rhap-(1-->3)- alpha -L-Rhap-(1-->2)- alpha-L-Rhap- (1-->2)- alpha-L-Rhap-(1-->2)- alpha-L-Rhap-(1-->. This structure is not identical to any of the previously described O-antigens of K. pneumoniae. The strong serological cross-reactivity with the Shigella flexneri serotype 6 O-antigen can most likely be attributed to the structural element alpha-L-Rhap-(1-->2)- alpha -L-Rhap present in the O-polysaccharide repeating unit of this serotype. Antiserum raised against the K. pneumoniae strain also agglutinated S. dysenteriae serotype 1 and strains of all different serotypes of S. flexneri. The Shigella strains contain the structural element alpha-L-Rhap-(1-->3)- alpha-L-Rhap in their O-antigen polysaccharides which may be responsible for the observed cross-reactivity.

The structures of the lipooligosaccharides (LOSs) from Haemophilus ducreyi ITM 5535 and ITM 3147 and a fresh clinical isolate, ACY1, have been investigated. Oligosaccharides were obtained from phenol-water-extracted LOS by mild acid hydrolysis and were studied by methylation analysis, fast atom bombardment and electrospray ionization mass spectrometry, and nuclear magnetic resonance spectroscopy. The major oligosaccharide obtained from all strains was a nonasaccharide with the structure beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-D-Galp-(1-->4)-D-a lpha-D-Hepp- (1-->6)-beta-D-Glcp-(1-->[L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp - (1-->3)]4)-L-alpha-D-Hepp-Kdo (Kdo stands for 3-deoxy-D-manno-octulosonic acid) and is thus identical to that identified as the major oligosaccharide in H. ducreyi ITM 2665 (E. K. H. Schweda, A. C. Sundström, L. M. Eriksson, J.A. Jonasson, and A. A. Lindberg, J. Biol. Chem. 269:12040-12048, 1994). Electrospray ionization mass spectrometry on O-deacylated LOS from H. ducreyi ITM 5535 obtained after treatment with anhydrous hydrazine gave evidence for the presence of a sialylated major compound, Neu5Ac alpha(2-->3)-beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-D-Gal p- (1-->4)-D-alpha-D-Hepp-(1-->6)-beta-D-Glcp-(1-->[L-alpha-D-Hepp -(1-->2)-L- alpha-D-Hepp-(1-->3)]4)-L-alpha-D-Hepp-Kdo(P)-O-deacylated lipid A (Neu5Ac stands for N-acetylneuraminic acid). However, an even larger oligosaccharide could be isolated from all strains as a minor component, viz., the undecasaccharide beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-d-Galp-(1-->4)-beta-D-glcNAcp-(1-->3)-beta-D-Galp-(1-->4)-D-alpha-D-Hepp-(1-->6)-beta-D-Glcp-(1-->[L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)]4-L-alpha-D-Hepp-Kdo, which represents an N-acetyl lactosamine disaccharide unit elongation of the LOS outer core. No Sialylation of this latter minor component undecasaccharide was detected.

A murine monoclonal antibody, MAHI 3 (immunoglobulin G2b), that is broadly reactive with Haemophilus influenzae lipopolysaccharides (LPSs) but nonreactive with all enterobacterial LPSs tested was generated by fusing mouse myeloma cells with spleen cells of BALB/c mice immunized with azide-killed H. influenzae RM.7004. MAHI 3 bound to all H. influenzae, all other human Haemophilus spp., all Bordetella pertussis and Bordetella parapertussis, and all Aeromonas spp. tested but not to any Neisseria or Moraxella catarrhalis strains, as determined by enzyme immunoassay, colony dot immunoblotting, and immunoblotting. In an inhibition enzyme immunoassay, MAHI 3 reacted with all 45 H. influenzae LPSs tested but not with the LPS from the rough mutant I69 Rd-/b+, which has only 3-deoxy-D-manno-octulosonic acid (P) [Kdo(P)] and lipid A. The antibody was not inhibited by H. influenzae lipid A or lipid-free polysaccharide isolated after mild acid hydrolysis. Only native LPSs show positive inhibitory activity, indicating that part of lipid A is involved in the binding of MAHI 3. From the results, it is indicated that the structural element recognized by MAHI 3 is Hep alpha 1-->2Hep alpha 1-->3Hep alpha 1-->Kdo together with part of lipid A, including the phosphate.

The capsular polysaccharide (CPS) of Vibrio cholerae O139 synonym Bengal, which is thought to carry determinants of O-specificity, was isolated by phenol/water extraction followed by delipidation of the contaminating lipopolysaccharide at pH 4.2 and gel-permeation chromatography. The CPS contained D-galactose, 3,6-dideoxy-L-xylo-hexose (colitose, Col), 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2,6-dideoxy-D-glucose (N-acetyl-D-quinovosamine, D-QuiNAc), D-galacturonic acid (D-GalA), and phosphate. The CPS was studied by NMR spectroscopy, methylation analysis, and selective degradations, including partial acid hydrolysis at pH 3.1 and dephosphorylation with aqueous 48% hydrofluoric acid, which both resulted in complete cleavage of Col. It was concluded that the CPS is built up of hexasaccharide repeating units containing inter alia D-galactose 4,6-cyclophosphate and having the following structure [structure: see text] These data basically confirm the structure of the V. cholerae CPS proposed on the basis of an NMR study [L. M. Preston et al. (1995) J. Bacteriol. 177, 835-838] and specify exactly the absolute configurations of the constituent monosaccharides and the position of the cyclic phosphate.