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A key challenge in the analytical assessment of therapeutic proteins is the comprehensive characterization of their higher-order structure (HOS). To directly assess HOS, a new type of assay is warranted. The most sensitive and detailed method for characterizing HOS is unquestionably nuclear magnetic resonance (NMR) spectroscopy. NMR spectroscopy provides direct information about the HOS at an atomic level, and with modern NMR spectrometers and improved pulse sequences, this has become feasible even on unlabeled proteins. Hence, NMR spectroscopy could be a very powerful tool for control of HOS following, for example, process changes resulting in structural changes, oxidation, degradation, or chemical modifications. We present a method for characterizing the HOS of therapeutic proteins by monitoring their methyl groups using 2D H, C-correlated NMR. We use a statistical model that compares the NMR spectrum of a given sample to a reference and results in one output value describing how similar the HOS of the samples are. This makes the overall result easy to interpret even for non-NMR experts. We show that the method is applicable to proteins of varying size and complexity (here up to ∼30 kDa) and that it is sufficiently sensitive for the detection of small changes in both primary and HOS. [ABSTRACT FROM AUTHOR]

1 H and 13 C NMR chemical shifts were accurately determined by consistent referencing for an extensive set of chemically synthesized branched α-glucan model compounds. The model compounds include anomerically fixed and reducing oligosaccharides ranging in size from isomaltose to a doubly branched decasaccharide. Both the 13 C1 chemical shift and the 13 C6 chemical shifts in α-(1→6) glycosidic bonds are strongly dependent on the chemical structure in the vicinity of the branch point, especially on the addition of glucopyranosyl units towards the non-reducing end of the backbone chain. The conformational sampling at the branch point of the branched α-glucan model compounds was experimentally probed with homo-nuclear scalar couplings. Substitution at O6 consistently increases the fraction of C6–O6 trans conformations, but to a lesser extent, if the attachment occurs at the reducing end residue. Increasingly complex structures in the vicinity of the branch point increase the population of the gauche – trans conformation of the C5–C6 bond. This population change is found to correlate with the 13 C6 chemical shift. [ABSTRACT FROM AUTHOR]

We report the repeating unit structures of the native capsular polysaccharides of Streptococcus pneumoniae serotypes 41A and 41F. Structural determinations yielded six carbohydrate units in the doubly branched repeating unit to give the following structure for serotype 41A: The structure determinations were motivated (1) by an ambition to help close the remaining gaps in S. pneumoniae capsular polysaccharide structures, and (2) by the attempt to derive functional annotations of carbohydrate active enzymes in the biosynthesis of bacterial polysaccharides from the determined structures. An activity present in 41F but not 41A is identified as an acetyltransferase acting on the rhamnopyranosyl sidechain E. The genes encoding the formation of the six glycosidic bonds in serogroup 41 were determined from the capsular polysaccharide structures of serotype 41A, 41F, and genetically related serotypes, in conjunction with corresponding genomic information and computational homology searches. In combination with complementary information, NMR spectroscopy considerably simplifies the functional annotation of carbohydrate active enzymes in the biosynthesis of bacterial polysaccharides. [ABSTRACT FROM AUTHOR]

The diversity of capsular polysaccharides of the bacterial pathogen Streptococcus pneumoniae leads to at least 91 different serotypes. While the genetic loci for capsular biosynthesis have been characterized for all serotypes, the determination of resultant polysaccharide structures remains incomplete. Here, we report the chemical structures of the capsular polysaccharides of serotypes 39, 42, and 47F from the genetic cluster 4, and discuss the structures in the context of structures from serologically and genetically related serotypes. Antigenic determinants can be approximated in this manner. The structure of the serotype 39 capsular polysaccharide is and has identical composition to the capsular polysaccharide 10A, but two different linkages. The serotype 42 structure closely resembles the genetically related serotype 35A, which does not contain residue A. The structure of the serotype 47F capsular polysaccharide is somewhat different from a recently determined structure from the same serogroup, while containing a structural motif that is reflected in serotype 35A and 42 capsular polysaccharide structures, thus explaining the cross-reactivity of serotype 47F with the typing serum 35a. [ABSTRACT FROM AUTHOR]

Highlights: [•] NMR spectroscopy is used to resolve cereal-derived carbohydrate mixtures in beer. [•] The 1H1–1H2 group is used as a structural reporter in two-dimensional spectra. [•] Structural assignments of more than 50 carbohydrate moieties are obtained. [•] Carbohydrates with suspected organoleptic and prebiotic function can be resolved. [•] Limiting enzyme activities can be deduced from motifs at glycan cleavage sites. [ABSTRACT FROM AUTHOR]

Highlights: [•] We assigned the NMR spectra’s of S. pneumoniae serotype 47A. [•] We established the structure of S. pneumoniae serotype 47A. [•] We compared the structural assignment with known serotypes. [•] We found a new substitution pattern for the β-Galf compared with known serotypes. [ABSTRACT FROM AUTHOR]

Abstract: The degradation of β-glucans from cereal cell walls is related to health benefits of whole grain foodstuffs and is a prominent cost in the production of bioethanol and in improving the filterability of malt-based beverages. Detailed assays of β-glucan degradation pathways by enzyme mixtures therefore promise to support the analysis of physiological and the optimization of technological processes. Physiological and biotechnological processes tend to occur in complex mixtures of catalysts and substrates and the development of advanced methodologies for mixture analysis has been attracting a great deal of attention. In situ detection of processes that involve carbohydrate synthesis and degradation encompasses the challenge of detecting monomer identities and linkage patterns, as well as enzymatic stereochemistry and specificity while resolving chemically similar reactants, challenges that are complicated in the additional detection of intermediate degradation steps. In the current study, we show that nuclear magnetic resonance (NMR) spectroscopy near the highest available spectrometer fields permits detailed real-time assays of the degradation of polymeric barley (1→3),(1→4)-β-glucan by commercial enzyme mixtures. Up to six different intermediates can be resolved and structurally assigned within a 0.07ppm chemical shift range using the anomeric 1H chemical signal in β-(1→3) glycosidic linkages as a structural reporter. More than 16 different glucopyranosyl spin systems are assigned to structural motifs in degradation fragments. The time course of intermediate emergence permits deciphering cleavage pathways and stereochemistry for up to four different enzyme catalyzed steps in situ. [Copyright &y& Elsevier]

Abstract: An increasingly detailed and realistic view of biological processes often hinges on atomic-level characterization of biomacromolecules and of the processes they are involved in, preferably under near-physiological conditions. Structure, degradation, and synthesis of glucose storage polymers have been studied for decades with a range of analytical tools, but the detailed in situ analysis has remained an analytical challenge. Here, we report the NMR assignment of different structural motifs in the β-limit dextrin from lintnerized maize starch as a branched α-glucan model system for starch, which is depleted of repetitive α-(1→4) glycosidic bonds at non-reducing ends but has the α-(1→6) branch points intact. By NMR spectroscopy at 18.7T magnetic field, we assign 12 discernible α-glucopyranosyl spin systems and identify them with different structural motifs. Amylolysis of the β-limit dextrin is directly followed by real-time NMR spectroscopy and four major cleavage products are identified and assigned to different branch point structures. Overall, these NMR assignments facilitate in situ assays under realistic conditions of substrate competition, transglycosylation, and product inhibition and shed light on chemical shift tendencies in different structural motifs of branched α-glucans. [Copyright &y& Elsevier]

Abstract: In the present study, the metabolic effects of heat and anoxic stress in myotubes from the mouse cell line C2C12 were investigated by using a combination of 13C, 1H, and 31P nuclear magnetic resonance (NMR) spectroscopy and enrichment with [13C]-glucose. Both the 13C and the 1H NMR spectra showed reduced levels of the amino acids alanine, glutamate, and aspartate after heat or anoxic stress. The decreases were smallest at 42°C, larger at 45°C, and most pronounced after anoxic conditions. In addition, in both the 1H and the 31P NMR spectra, decreases in the high-energy phosphate compounds adenosine triphosphate and phosphocreatine with increasing severity of stress were identified. At anoxic conditions, an increase in 13C-labeled lactate and appearance of glycerol-3-phosphate were observed. Accumulation of lactate and glycerol-3-phosphate is in agreement with a shift to anaerobic metabolism due to inhibition of the aerobic pathway in the mitochondria. Conversely, lower levels of unlabeled (12C) lactate were apparent at increasing severity of stress, which indicate that lactate is released from the myotubes to the medium. In conclusion, the metabolites identified in the present study may be useful markers for identifying severity of stress in muscles. [Copyright &y& Elsevier]

Abstract: A phytochemical re-investigation of Tragopogon porrifolius L. (Asteraceae) yielded (7S,15S)-2,4,12-trihydroxy-7-(4-hydroxyphenyl)-10-methoxy-15-(4-methoxyphenyl)-7,8,15,16-tetrahydrodibenzo[c,i][1,7]dioxacyclododecine-5,13-dione, named tragoponol, a dimeric dihydroisocoumarin. The compound, which represents the first of its kind, is comprised of the open-chained forms of two different mono-methoxylated dihydroisocoumarin moieties, scorzocreticin and hongkongenin, which are connected via two ester bonds to form a macrolide with two lactone moieties featuring a 12-membered ring. The structure of the nearly symmetrical compound was established by HR mass spectrometry, CD measurements, and extensive 1D and 2D NMR experiments. [Copyright &y& Elsevier]

Abstract: The new NMR experiments 3D H2BC and clean HMBC are explored for challenging applications to a complex carbohydrate at natural abundance of 13C. The 3D H2BC experiment is crucial for sequential assignment as it yields heteronuclear one- and two-bond together with COSY correlations for the 1H spins, all in a single spectrum with good resolution and non-informative diagonal-type peaks suppressed. Clean HMBC is a remedy for the ubiquitous problem of strong coupling induced one-bond correlation artifacts in HMBC spectra of carbohydrates. Both experiments work well for one of the largest carbohydrates whose structure has been determined by NMR, not least due to the enhanced resolution offered by the third dimension in 3D H2BC and the improved spectral quality due to artifact suppression in clean HMBC. Hence these new experiments set the scene to take advantage of the sensitivity boost achieved by the latest generation of cold probes for NMR structure determination of even larger and more complex carbohydrates in solution. [Copyright &y& Elsevier]

Abstract: Nuclear magnetic resonance (NMR)–based metabonomics was carried out on plasma samples from a total of seventy-five 17-year–old Danes to investigate the impact of key parameters such as sex, height, weight, and body mass index on the plasma metabolite profile in a normal, healthy population. Principal component analysis identified sex to have a large impact on the NMR plasma metabolome, whereas no apparent effects of height, weight, and body mass index were found. Partial least square regression discriminant analysis and quantification of relative metabolite concentrations by integration of NMR signals revealed that the sex effect included differences in plasma lipoproteins (mainly high-density lipoprotein), glucose, choline, and amino acid content. Accordingly, the present study suggests a higher lipid synthesis in young women than young men and a higher protein turnover in young men compared with women. Data on plasma content of triglyceride, lipoprotein fractions, and cholesterol at an age of 9 months were available for selected individuals (n = 40); and partial least square regressions revealed correlations between these infant parameters and the NMR plasma metabolome at an age of 17 years. In conclusion, the present study demonstrates the feasibility of NMR-based metabonomics for obtaining a deeper insight into interindividual differences in metabolism and for exploring relationships between parameters measured early in life and metabolic status at a later stage. [Copyright &y& Elsevier]

Abstract: Complete assignment of 1H and 13C NMR of six hexagalactopyranuronic acids with varying degree and pattern of methyl esterification is reported. The NMR experiments were run at room temperature using approximately 2mg of sample making this method convenient for studying the structure of homogalacturonan oligosaccharides. [Copyright &y& Elsevier]

Abstract: The secondary cell wall polymer (SCWP) from Geobacillus stearothermophilus PV72/p2, which is involved in the anchoring of the surface-layer protein to the bacterial cell wall layer, is composed of 2-amino-2-deoxy- and 2-acetamido-2-deoxy-d-glucose, 2-acetamido-2-deoxy-d-mannose, and 2-acetamido-2-deoxy-d-mannuronic acid. The primary structure of the acid-degraded polysaccharide—liberated by HF-treatment from the cell wall—was determined by high-field NMR spectroscopy and mass spectrometry using N-acetylated and hydrolyzed polysaccharide derivatives as well as Smith-degradation. The polysaccharide was shown to consist of a tetrasaccharide repeating unit containing a pyruvic acid acetal at a side-chain 2-acetamido-2-deoxy-α-d-mannopyranosyl residue. Substoichiometric substitutions of the repeating unit were observed concerning the degree of N-acetylation of glucosamine residues and the presence of side-chain linked 2-acetamido-2-deoxy-β-d-glucopyranosyl units: Display Omitted [Copyright &y& Elsevier]

Abstract: Two galactofuranomannans, Ths-4 and Ths-5, were isolated from the lichen, Thamnolia vermicularis var. subuliformis, using ethanol fractionation and anion-exchange and size-exclusion chromatography. The average molecular weights of Ths-4 and Ths-5 were estimated to be 19 and 200kDa, respectively. Structural characterisation of Ths-4, Ths-5 and their partially hydrolysed derivatives was performed by methanolysis and methylation analysis. The intact and partially hydrolysed Ths-4 was further analysed using NMR spectroscopy (1D, COSY, NOESY, TOCSY, HSQC and HMBC). According to the data obtained, the heteroglycans Ths-4 and Ths-5 have similar structures, but have large differences in molecular weight. The structure is composed of 3-O-linked and 5-O-linked galactofuranosyl chains linked to a mannan core. The mannan core consists of a main chain of α-(1→6)-linked mannopyranosyl residues, substituted at O-2 with either a single α-mannopyranosyl unit or an α-Manp-(1→2)-α-Manp-(1→2)-α-Manp group in the ratio of approximately 1:3, respectively. The polysaccharides have idealised repeating blocks as is shown. [Copyright &y& Elsevier]

The structures of the lipopolysaccharide (LPS) core and O antigena of Bordetella bronchiseptica and Bordetella parapertussis are known, but how these two regions are linked to each other had not been determined. We have studied LPS from several strains of these microorganisms to determine the complete carbohydrate structure of the LPS. LPS was analyzed using different chemical degradations, NMR spectroscopy, and mass spectrometry. This identified a novel pentasaccharide fragment that links the O chain to the core in all the LPS studied. In addition, although the O chain of these bacteria was reported as a homopolymer of 1,4-linked 2,3-diacetamido-2,3- dideoxy-α-galacturonic acid, we discovered that the polymer contains several amidated uronic acids, the number of which varies between strains. These new data describe the complete structure of the LPS carbohydrate backbone for both Bordetella species and help to explain the complex genetics of LPS biosynthesis in these bacteria. [ABSTRACT FROM AUTHOR]

Abstract: It is demonstrated that the H2BC NMR pulse sequence (J. Am. Chem. Soc. 2005, 127, 6154, Magn. Reson. Chem. 2005, 43, 971–974) offers unambiguous assignments and significant simplification of NMR spectra of large and complex carbohydrates compared to other techniques for the establishment of correlations over more than one bond. H2BC almost exclusively correlates protons and proton-bearing carbon spins separated by two covalent bonds and is independent of occasionally vanishing 2 J CH coupling constants, which alleviates the problem of missing two-bond correlations in HMBC spectra. H2BC also solves the problem of distinguishing two- and three-bond correlations in HSQC–TOCSY or HMBC. It is a further asset of H2BC that the experiment is significantly shorter than HMBC and HSQC–TOCSY, and hence less sensitive to transverse relaxation. The H2BC experiment is demonstrated on an approximately 30-residue oligosaccharide from Francisella victoria. [Copyright &y& Elsevier]

Art v 1, the major allergen of mugwort (Artemisia vulgaris) pollen contains galactose and arabinose. As the sera of some allergic patients react with natural but not with recombinant Art v 1 produced in bacteria, the glycosylation of Art v 1 may play a role in IgE binding and human allergic reactions. Chemical and enzymatic degradation, mass spectrometry, and 800 MHz 1H and 13C nuclear magnetic resonance spectroscopy indicated the proline-rich domain to be glycosylated in two ways. We found a large hydroxyproline-linked arabinogalactan composed of a short βl,6-galactan core, which is substituted by a variable number (5–28) of α-arabinofuranose residues, which form branched side chains with 5-, 2,5-, 3,5-, and 2,3,5-substituted arabinoses. Thus, the design of the Art v 1 polysaccharide differs from that of the well known type II arabinogalactans, and we suggest it be named type III arabinogalactan. The other type of glycosylation was formed by single (but adjacent) β-arabinofuranoses linked to hydroxyproline. In contrast to the arabinosylation of Ser-Hyp4 motifs in other hydroxyproline-rich glycoproteins, such as extensins or solanaceous lectins, no oligo-arabinosides were found in Art v 1. Art v 1 and parts thereof produced by alkaline degradation, chemical deglycosylation, proteolytic degradation, and/or digestion with α-arabinofuranosidase were used in enzyme-linked immunosorbent assay and immunoblot experiments with rabbit serum and with the sera of patients. Although we could not observe antibody binding by the polysaccharide, the single hydroxyproline-linked β-arabinose residues appeared to react with the antibodies. Mono-β-arabinosylated hydroxyproline residues thus constitute a new, potentially cross-reactive, carbohydrate determinant in plant proteins. [ABSTRACT FROM AUTHOR]

Abstract: A phytochemical investigation of Podospermum laciniatum (L.) DC. (Asteraceae) yielded the new quinic acid derivative podospermic acid (1,3,5-tridihydrocaffeoylquinic acid), which was named after the genus it was isolated from. The structure was established by HR mass spectrometry and extensive 1D and 2D NMR spectroscopy. Podospermic acid is the first naturally occurring dihydrocaffeoylquinic acid derivative. The chemosystematic impact and the radical scavenging activity of the new compound are discussed briefly. [Copyright &y& Elsevier]

The yellow brain mushroom Tremella mesenterica possesses a wide spectrum of medicinal properties, including immunostimulating, protecting against radiation, antidiabetic, anti-inflammatory, hypocholesterolemic, hepatoprotective, and antiallergic effects. A unique feature of T. mesenterica is that most of the above mentioned medicinal properties depend on glucuronoxylomannan (GXM) contained in fruiting bodies or produced in pure culture conditions. We developed a new strain of T. mesenterica CBS 101939, which grows in submerged culture and offers superior yields of one-cell biomass rich in exocellular heteropolysaccharide GXM. The structure of the GXM was analyzed by NMR spectroscopy and chemical methods. The polysaccharide has a defined repeating unit structure, which is O-acetylated at several points:These results differ from previously published structure of Tremella extracellular polysaccharides, where mannan backbone was believed to be randomly glycosylated with xylan chains of different length. [Copyright &y& Elsevier]

The structure of the LPS from Serratia marcescens serotype O19 was investigated. Deamination of the LPS released the O-chain polysaccharide together with a fragment of the core oligosaccharide. The following structure of the product was determined by NMR spectroscopy, mass spectrometry, and chemical methods:The main polymer consists of a repeating disaccharide V-U and is present on average of 18 units per chain as estimated by integration of signals in the NMR spectra. The residue O corresponds to the primer, which initiates biosynthesis of the O-chain, and an oligomer of a disaccharide R-S is an insert between the primer and the main polymer. The polysaccharide has a β-Kdo residue at the non-reducing end, a feature similar to that observed previously in the LPS from Klebsiella O12. [Copyright &y& Elsevier]

Abstract: Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α-d-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of α-d-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming β-glucosyl disaccharides with β-(1→4)- regioselectivity from five monosaccharides as well as branched β-glucosyl trisaccharides with β-(1→4)-regioselectivity from three (1→6)-linked disaccharides. CtCDP showed strict β-(1→4)-regioselectivity and catalysed linear chain extension of the three β-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two β-glucosyl oligosaccharide product series to represent novel compounds, i.e. β-d-glucopyranosyl-[(1→4)-β-d-glucopyranosyl]n-(1→2)-d-glucopyranose, and β-d-glucopyranosyl-[(1→4)-β-d-glucopyranosyl]n-(1→3)-d-glucopyranose (n = 1–7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the C1 hydroxyl of β-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of C1 substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637–Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using α-d-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides. [Copyright &y& Elsevier]

Abstract: Gb3 and iGb3 are physiologically important trihexosylceramides with a terminal α-d-Galp-(1→4)-β-d-Galp- and α-d-Galp-(1→3)-β-d-Galp sequence, respectively. In particular iGb3 is attracting considerable attention as it is believed to serve as a ligand for natural killer T cells. Whether or not iGb3 is present in humans and which enzyme might be responsible for its synthesis is at present a matter of lively debate. In the current investigation we evaluated human blood group B galactosyltransferase (GTB) for its ability to catalyze the formation of iGb3 from lactosylceramide and UDP-Galp. GTB is a retaining glycosyltransferase that in vivo catalyzes the transfer of galactose from UDP-Galp donors to OH-3 of Galp on the H-antigen (α-l-Fucp-(1→2)-β-d-Galp) acceptor forming the blood group B antigen. GTB tolerates modifications in donor and acceptor substrates and its ability to accept lactosides as acceptors makes it a possible candidate for iGb3 production in humans. For comparison iGb3 and Gb3 were also synthesized from the same acceptor using an α-(1→3)- and α-(1→4)-specific galactosyltransferase, respectively. All the enzymes tested catalyzed the desired reactions. Product characterization by NMR analysis clearly differentiated between the α-Galp-(1→3)-Galp and α-Galp-(1→4)-Galp product, with the GTB product being identical to that of the α-(1→3)-GalT-catalyzed reaction. The rate of transfer by GTB however was very low, only 0.001% of the rate obtained with a good substrate, H antigen disaccharide (octyl α-l-Fucp-(1→2)-β-d-Galp). This is too low to account for the possible formation of the iGb3 structure in humans in vivo. [Copyright &y& Elsevier]

Abstract: Medium dependant production and the structure elucidation of corymbiferone (1) from the fungus Penicillium hordei grown on oatmeal and macerated tulip, yellow onion and red onion agars are reported. Compound 1 possesses an unusual oxygenated aromatic structure with a lactone bridge preventing full aromatization of the molecule, resulting in several unusual chemical shifts. [Copyright &y& Elsevier]

A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation. [ABSTRACT FROM AUTHOR]

There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galβ1-4GlcNAcβ- tetramethylrhodamine (LacNAc-TMR (Galβ1-4GlcNAcβ (CH2)8- CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcβ1-4GlcNAcβ-TMR), LacNAc-TMR, and LacNAcβ1-6LacNAcβ-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the β1-6 bond in LacNAcβ1-6LacNAcβ-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis. [ABSTRACT FROM AUTHOR]

Abstract: The bark of Cola cordifolia used in Malian traditional medicine contains unusual types of polysaccharides with immunomodulating activities. We report for the first time on the structure of a polymer designated CC1P1 having the repeating structure [2→)[α-d-Gal(1→3)]α-l-Rha(1→4)α-d-GalA(1→] as determined by NMR and GC/MS. α-Linked Gal is unusual in pectins. The M w of 135kDa was determined by SEC-MALLS. CC1P2 (1400kDa), another polymer, having the same backbone, but this was substituted with α-4-OMe-GlcA, α-2-OMe-Gal and α-Gal as terminal units. CC1P1 shows a high complement-fixing activity, IC50 being 2.2 times lower than the positive pectin control PMII (IC50 appr. 71μg/mL) while IC50 of CC1P2 is 1.8 times lower. The simple structure of CC1P1 did not activate macrophages, while CC1P2 (100μg/mL) showed the same potency as the positive controls PMII (100μg/mL) and LPS (500ng/mL). No cytotoxicity was detected. [Copyright &y& Elsevier]

Abstract: Six new meroterpenoid compounds, hesseltins B–G, were isolated from Penicillium species along with the previously described hesseltin A. A further 14 compounds, which turned out to be photoisomers, were detected during purification and were subsequently isolated. The structures of these analogues were elucidated by comparison of their NMR spectral data with that of hesseltin A. None of the new hesseltins showed antiviral activity in a Herpes simplex virus type 2 assay. [ABSTRACT FROM AUTHOR]

Art v 1, the major pollen allergen of the composite plant mugwort (Artemisia vulgaris) has been identified recently as a thionin- like protein with a bulky arabinogalactan-protein moiety. A close relative of mugwort, ragweed (Ambrosia artemisiifolia) is an important allergen source in North America, and, since 1990, ragweed has become a growing health concern in Europe as well. Weed pollen-sensitized patients demonstrated IgE reactivity to a ragweed pollen protein of apparently 29-31 kDa. This reaction could be inhibited by the mugwort allergen Art v 1. The purified ragweed pollen protein consisted of a 57-amino acid-long defensin-like domain with high homology to Art v 1 and a C-terminal proline-rich domain. This part contained hydroxyproline- linked arabinogalactan chains with one galactose and 5 to 20 and more α-arabinofuranosyl residues with some β-arabinoses in terminal positions as revealed by high field NMR. The ragweed protein contained only small amounts of the single hydroxyproline-linked β-arabinosyl residues, which form an important IgE binding determinant in Art v 1. cDNA clones for this protein were obtained from ragweed flowers. Immunological characterization revealed that the recombinant ragweed protein reacted with > 30% of the weed pollen allergic patients. Therefore, this protein from ragweed pollen constitutes a novel important ragweed allergen and has been designated Amb a 4. [ABSTRACT FROM AUTHOR]

Abstract: Protection against pneumococcal disease is thought to be mediated primarily by antibodies that are opsonic [Musher DM, Chapman AJ, Goree A, Jonsson S, Briles D, Baughn RE. Natural and vaccine-related immunity to Streptococcus pneumoniae. J Infect Dis 1986;154(2):245–56]. Pneumococcal capsular polysaccharide (CPS) is immunogenic and induces type-specific protective immunity. For convenience, the protective capacity of serum antibodies is often evaluated by the measurement of antibody titers in an ELISA test. The pneumococcal capsular polysaccharide (CPS) used in ELISA contains several impurities; these include about 5% by weight of teicholic acid (CWPS) and the cholin binding protein, pneumococcal surface protein A (PspA) [Sorensen UB, Henrichsen J. C-polysaccharide in a pneumococcal vaccine. Acta Pathol Microbiol Immunol Scand C 1984;92(6):351–6; Yu J, Briles DE, Englund JA, Hollingshead SK, Glezen WP, Nahm MH. Immunogenic protein contaminants in pneumococcal vaccines. J Infect Dis 2003;187(6):1019–23]. All individuals have antibodies to CWPS possible as a result of early exposure to pneumococci, Streptocuccus mitis and Streptocuccus oralis [Bergstrom N, Jansson PE, Kilian M, Skov Sorensen UB. Structures of two cell wall-associated polysaccharides of a Streptococcus mitis biovar 1 strain. A unique teichoic acid-like polysaccharide and the group O antigen which is a C-polysaccharide in common with pneumococci. Eur J Biochem 2000;267(24):7147–57. ]. The concentration of the CWPS antibodies in non-immunized individuals often exceeds the concentration of the serotype-specific pneumococcal antibodies. Therefore, the pneumococcal ELISA requires an adsorption step to remove the unprotective CWPS antibodies [Konradsen HB, Sorensen UB, Henrichsen J. A modified enzyme-linked immunosorbent assay for measuring type-specific anti-pneumococcal capsular polysaccharide antibodies. J Immunol Meth 1993;164(1):13–20. ; Concepcion N, Frasch CE. Evaluation of previously assigned antibody concentrations in pneumococcal polysaccharide reference serum 89SF by the method of cross-standardization. Clin Diagn Lab Immunol 1998;5(2):199–204. ; Kayhty H, Ahman H, Ronnberg PR, Tillikainen R, Eskola J. Pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine is immunogenic in infants and children. J Infect Dis 1995;172(5):1273–8. ; Koskela M. Serum antibodies to pneumococcal C polysaccharide in children: response to acute pneumococcal otitis media or to vaccination. Pediatr Infect Dis J 1987;6 (6):519–26. ]. Recently a new pneumococcal CPS ELISA was recommended with an extra serum absorption step with 22F CPS to remove antibodies against an extra unknown common cross-reactive component. The aim of this study was to characterize the active component in the 22F capsule. A non-capsulated pneumococci was prepared from a 22F capsulated pneumococci. The cell wall polysaccharide (CWPS2) purified from this pneumococci has a better adsorption potential than 22F capsule in the pneumococci ELISA. Structure characterization of the commercial available CWPS and CWPS2 was done by nuclear magnetic resonance (NMR). The NMR results showed that commercial CWPS had one phosporylcholine per sugar repeat while the CWPS2 had two phosporylcholine per sugar repeat explaining an immunological difference between the two variants of CWPS. In addition the LicD2 gene responsible for the attachment of the second cholin in the CWPS tetra sugar repeat was inactive in the strain used for purifying the commercial CWPS but active in the strain expressing CWPS2. [Copyright &y& Elsevier]

Abstract: The oxidation of spermidine or homospermidine with bovine serum amine oxidase (BSAO) was monitored in situ, using proton nuclear magnetic resonance spectroscopy in water with 10% D2O. NMR assignments were performed by spin decoupling and COSY spectra or by comparison with data from synthetic aminoaldehydes. The results represent the first in situ characterisation of the highly reactive aminoaldehydes and showed oxidation at the N1 amino group of spermidine and homospermidine. Comparison of homospermidine with a variety of substrates revealed that among straight chain di- and polyamines both an aminopropyl group and two primary amino groups separated by seven (norspermidine) or eight (spermidine) carbon atoms were required for optimal substrate ability. However, highest activity was seen with the substrate N-(4-aminobutyl)hexahydropyrimidine, showing that the substrate channel of BSAO has a dual substrate preference, with moderately bulky substituents at the distal end of a diamine contributing equally well as an alkyl amino group. Cytotoxic investigations of a variety of substrates for BSAO, confirmed previous results, that cytotoxicity is primarily linked to polyamines encompassing the aminopropyl moiety. No acrolein was observed at any time during the oxidation showing that it reacts very fast with available amino groups forming a variety of derivatives. [Copyright &y& Elsevier]