Normal Neurodevelopment and Fertility in Juvenile Male Rats Exposed to Polyethylene Glycol Following Dosing With PEGylated rFIX (Nonacog Beta Pegol, N9-GP): Evidence from a 10-Week Repeat-Dose Toxicity Study.

N9-GP/Rebinyn®/Refixia® is an approved PEGylated (polyethylene glycol-conjugated) recombinant human factor IX intended for prophylactic and/or on-demand treatment in adults and children with haemophilia B. A juvenile neurotoxicity study was conducted in male rats to evaluate effects on neurodevelopment, sexual maturation, and fertility following repeat-dosing of N9-GP. Male rats were dosed twice weekly from Day 21 of age with N9-GP or vehicle for 10 weeks, followed by a dosing-free recovery period for 13 weeks and terminated throughout the dosing and recovery periods. Overall, dosing N9-GP to juvenile rats did not result in any functional or pathological effects, as measured by neurobehavioural/neurocognitive tests, including motor activity, sensory function, learning and memory as well as growth, sexual maturation, and fertility. This was further supported by the extensive histopathologic evaluation of brain tissue. Exposure and distribution of polyethylene glycol was investigated in plasma, choroid plexus, cerebrospinal fluid, and brain sections. PEG did not cross the blood brain barrier and PEG exposure did not result in any effects on neurodevelopment. In conclusion, dosing of N9-GP to juvenile rats did not identify any effects on growth, sexual maturation and fertility, clinical and histological pathology, or neurodevelopment related to PEG exposure and supports the prophylactic use of N9-GP in children.

A diverse range of bacterial and eukaryotic chitinases hydrolyzes the LacNAc (Galß1-4GlcNAc) and LacdiNAc (GalNAcß1-4GlcNAc) motifs found on vertebrate and insect cells.

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 Galß1-4GlcNAcß-tetramethylrhodamine (LacNAc-TMR (Galß1-4GlcNAcß(CH2)8CONH(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 (GalNAcß1-4GlcNAcß-TMR), LacNAc-TMR, and LacNAcß1-6LacNAcß-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 ß1-6 bond in LacNAcß1-6LacNAcß-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.

An efficient arabinoxylan-debranching α-L-arabinofuranosidase of family GH62 from Aspergillus nidulans contains a secondary carbohydrate binding site.

An α-L-arabinofuranosidase of GH62 from Aspergillus nidulans FGSC A4 (AnAbf62A-m2,3) has an unusually high activity towards wheat arabinoxylan (WAX) (67 U/mg; k cat = 178/s, K m = 4.90 mg/ml) and arabinoxylooligosaccharides (AXOS) with degrees of polymerisation (DP) 3-5 (37-80 U/mg), but about 50 times lower activity for sugar beet arabinan and 4-nitrophenyl-α-L-arabinofuranoside. α-1,2- and α-1,3-linked arabinofuranoses are released from monosubstituted, but not from disubstituted, xylose in WAX and different AXOS as demonstrated by NMR and polysaccharide analysis by carbohydrate gel electrophoresis (PACE). Mutants of the predicted general acid (Glu(188)) and base (Asp(28)) catalysts, and the general acid pK a modulator (Asp(136)) lost 1700-, 165- and 130-fold activities for WAX. WAX, oat spelt xylan, birchwood xylan and barley ß-glucan retarded migration of AnAbf62A-m2,3 in affinity electrophoresis (AE) although the latter two are neither substrates nor inhibitors. Trp(23) and Tyr(44), situated about 30 Å from the catalytic site as seen in an AnAbf62A-m2,3 homology model generated using Streptomyces thermoviolaceus SthAbf62A as template, participate in carbohydrate binding. Compared to wild-type, W23A and W23A/Y44A mutants are less retarded in AE, maintain about 70 % activity towards WAX with K i of WAX substrate inhibition increasing 4-7-folds, but lost 77-96 % activity for the AXOS. The Y44A single mutant had less effect, suggesting Trp(23) is a key determinant. AnAbf62A-m2,3 seems to apply different polysaccharide-dependent binding modes, and Trp(23) and Tyr(44) belong to a putative surface binding site which is situated at a distance of the active site and has to be occupied to achieve full activity.

Fast and accurate quantitation of glucans in complex mixtures by optimized heteronuclear NMR spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy is a widely used technique for mixture analysis, but it has shortcomings in resolving carbohydrate mixtures due to the narrow chemical shift range of glycans in general and fragments of homopolymers in particular. Here, we suggest a protocol toward fast spectroscopic glycan mixture analysis. We show that a plethora of oligosaccharides comprising only α-glucopyranosyl residues can be resolved into distinct quantifiable signals with NMR experiments that are substantially faster than chromatographic runs. Conceptually, the approach fully exploits the narrow line widths of glycans (ν1/2 < 3 Hz) in the (13)C spectral dimension while disregarding superfluous spectral information in compound identification and quantitation. The acetal (H1C1) groups suffice to spectroscopically resolve ∼20 different starch fragments in optimized (1)H-(13)C NMR with a narrow (13)C spectral width (3 ppm) that allows sampling the indirect (13)C dimension at high resolution within 15 min. Rapid quantitations by high-resolution NMR data are achieved for glycans at concentrations as low as 10 µg/mL. For validation, comparisons were made with quantitations obtained by more time-consuming chromatographic methods and yielded coefficients of determination (R(2)) above 0.99.

Time-resolved in-situ observation of starch polysaccharide degradation pathways.

Analytical challenges in the direct time-resolved observation of starch metabolism have been addressed by using optimized multidimensional NMR experiments. Starch provides the main source of human dietary energy intake and is a raw material for beverage and renewable fuel production. Use of direct in situ observations of starch remodeling pathways could facilitate our understanding and control of processes of biotechnological, medical, and environmental relevance. Processes involving starch synthesis or degradation are difficult to monitor directly in aqueous solution, however, because starch consists of glucopyranosyl homopolymers that are built up from and degraded into structurally similar fragments that yield only small signal dispersion in optical and NMR spectroscopy. By focusing on acetal groups only, (1) H,(13) C HSQC experiments sampling narrow spectral windows in the highly resolved (13) C dimension have been employed in order to observe the amylopectin cleavage pathway in real time with a temporal resolution of 150 s. Quantifiable signals for more than 15 molecular species emerging during starch fragmentation by human saliva have been resolved and tracked over time in this manner. Altered accumulation of intermediates in the digestion of amylopectin in the presence of black tea acting as an effector have been monitored.

A new allergen from ragweed (Ambrosia artemisiifolia) with homology to art v 1 from mugwort.

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 alpha-arabinofuranosyl residues with some beta-arabinoses in terminal positions as revealed by high field NMR. The ragweed protein contained only small amounts of the single hydroxyproline-linked beta-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.

Characterization of a novel Salmonella Typhimurium chitinase which hydrolyzes chitin, chitooligosaccharides and an N-acetyllactosamine conjugate.

Salmonella contain genes annotated as chitinases; however, their chitinolytic activities have never been verified. We now demonstrate such an activity for a chitinase assigned to glycoside hydrolase family 18 encoded by the SL0018 (chiA) gene in Salmonella enterica Typhimurium SL1344. A C-terminal truncated form of chiA lacking a putative chitin-binding domain was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3) with an N-terminal (His)(6) tag. The purified enzyme hydrolyzes 4-nitrophenyl N,N'-diacetyl-ß-D-chitobioside, 4-nitrophenyl ß-D-N,N',N″-triacetylchitotriose and carboxymethyl chitin Remazol Brilliant Violet but does not act on 4-nitrophenyl N-acetyl-ß-D-glucosaminide, peptidoglycan or 4-nitrophenyl ß-D-cellobioside. Enzyme activity was also characterized by directly monitoring product formation using (1)H-nuclear magnetic resonance which showed that chitin is a substrate with the release of N,N'-diacetylchitobiose. Hydrolysis occurs with the retention of configuration and the enzyme acts on only the ß-anomers of chitooligosaccharide substrates. The enzyme also released N-acetyllactosamine disaccharide from Galß1 → 4GlcNAcß-O-(CH(2))(8)CONH(CH(2))(2)NHCO-tetramethylrhodamine, a model substrate for LacNAc terminating glycoproteins and glycolipids.

Improved characterization of nod factors and genetically based variation in LysM Receptor domains identify amino acids expendable for nod factor recognition in Lotus spp.

Formation of functional nodules is a complex process depending on host-microsymbiont compatibility in all developmental stages. This report uses the contrasting symbiotic phenotypes of Lotus japonicus and L. pedunculatus, inoculated with Mesorhizobium loti or the Bradyrhizobium sp. (Lotus), to investigate the role of Nod factor structure and Nod factor receptors (NFR) for rhizobial recognition, infection thread progression, and bacterial persistence within nodule cells. A key contribution was the use of 800 MHz nuclear magnetic resonance spectroscopy and ultrahigh-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry for Nod factor analysis. The Nod factor decorations at the nonreducing end differ between Bradyrhizobium sp. (Lotus) and M. loti, and the NFR1/NFR5 extracellular regions of L. pedunculatus and L. japonicus were found to vary in amino acid composition. Genetic transformation experiments using chimeric and wild-type receptors showed that both receptor variants recognize the structurally different Nod factors but the later symbiotic phenotype remained unchanged. These results highlight the importance of additional checkpoints during nitrogen-fixing symbiosis and define several amino acids in the LysM domains as expendable for perception of the two differentially carbamoylated Nod factors.

Metabolomic signatures of inbreeding at benign and stressful temperatures in Drosophila melanogaster.

While the population genetics of inbreeding is fairly well understood, the effects of inbreeding on the physiological and biochemical levels are not. Here we have investigated the effects of inbreeding on the Drosophila melanogaster metabolome. Metabolite fingerprints in males from five outbred and five inbred lines were studied by nuclear magnetic resonance spectroscopy after exposure to benign temperature, heat stress, or cold stress. In both the absence and the presence of temperature stress, metabolite levels were significantly different among inbred and outbred lines. The major effect of inbreeding was increased levels of maltose and decreased levels of 3-hydroxykynurenine and a galactoside [1-O-(4-O-(2-aminoethyl phosphate)-beta-d-galactopyranosyl)-x-glycerol] synthesized exclusively in the paragonial glands of Drosophila species, including D. melanogaster. The metabolomic effect of inbreeding at the benign temperature was related to gene expression data from the same inbred and outbred lines. Both gene expression and metabolite data indicate that fundamental metabolic processes are changed or modified by inbreeding. Apart from affecting mean metabolite levels, inbreeding led to an increased between-line variation in metabolite profiles compared to outbred lines. In contrast to previous observations revealing interactions between inbreeding and environmental stress on gene expression patterns and life-history traits, the effect of inbreeding on the metabolite profile was similar across the different temperature treatments.

Higher-Order Structure Characterization of Pharmaceutical Proteins by 2D Nuclear Magnetic Resonance Methyl Fingerprinting.

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.

Structural characterization of the acid-degraded secondary cell wall polymer of Geobacillus stearothermophilus PV72/p2.

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-alpha-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-beta-D-glucopyranosyl units: [Formula: see text].

Characterization of the lipopolysaccharide and beta-glucan of the fish pathogen Francisella victoria.

Lipopolysaccharide (LPS) and beta-glucan from Francisella victoria, a fish pathogen and close relative of highly virulent mammal pathogen Francisella tularensis, have been analyzed using chemical and spectroscopy methods. The polysaccharide part of the LPS was found to contain a nonrepetitive sequence of 20 monosaccharides as well as alanine, 3-aminobutyric acid, and a novel branched amino acid, thus confirming F. victoria as a unique species. The structure identified composes the largest oligosaccharide elucidated by NMR so far, and was possible to solve using high field NMR with cold probe technology combined with the latest pulse sequences, including the first application of H2BC sequence to oligosaccharides. The non-phosphorylated lipid A region of the LPS was identical to that of other Francisellae, although one of the lipid A components has not been found in Francisella novicida. The heptoseless core-lipid A region of the LPS contained a linear pentasaccharide fragment identical to the corresponding part of F. tularensis and F. novicida LPSs, differing in side-chain substituents. The linkage region of the O-chain also closely resembled that of other Francisella. LPS preparation contained two characteristic glucans, previously observed as components of LPS preparations from other strains of Francisella: amylose and the unusual beta-(1-6)-glucan with (glycerol)2phosphate at the reducing end.

Structural characterisation of novel lichen heteroglycans by NMR spectroscopy and methylation analysis.

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 200 kDa, 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 alpha-(1-->6)-linked mannopyranosyl residues, substituted at O-2 with either a single alpha-mannopyranosyl unit or an alpha-Manp-(1-->2)-alpha-Manp-(1-->2)-alpha-Manp group in the ratio of approximately 1:3, respectively. The polysaccharides have idealised repeating blocks as is shown.

H2BC: a new technique for NMR analysis of complex carbohydrates.

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.

Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin.

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.

NMR characterization of chemically synthesized branched α-dextrin model compounds.

1H and 13C 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 13C1 chemical shift and the 13C6 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 13C6 chemical shift.

Hesseltin A, a novel antiviral metabolite from Penicillium hesseltinei.

[structure: see text] Hesseltin A 1, a novel compound of mixed polyketide-terpenoid origins was isolated from the filamentous fungus Penicillium hesseltinei. The structure and stereochemistry were determined from extensive one- and two-dimensional NMR and mass spectral data.

The structure of the glucuronoxylomannan produced by culinary-medicinal yellow brain mushroom (Tremella mesenterica Ritz.:Fr., Heterobasidiomycetes) grown as one cell biomass in submerged culture.

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: [structure: see text]. 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.

The structure of the polysaccharide part of the LPS from Serratia marcescens serotype O19, including linkage region to the core and the residue at the non-reducing end.

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: [carbohydrate structure: see text] 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 beta-Kdo residue at the non-reducing end, a feature similar to that observed previously in the LPS from Klebsiella O12.

(1)H NMR spectroscopy for profiling complex carbohydrate mixtures in non-fractionated beer.

A plethora of biological and biotechnological processes involve the enzymatic remodelling of carbohydrates in complex mixtures whose compositions affect both the processes and products. In the current study, we employed high-resolution (1)H NMR spectroscopy for the analysis of cereal-derived carbohydrate mixtures as exemplified on six beer samples of different styles. Structural assignments of more than 50 carbohydrate moieties were obtained using (1)H1-(1)H2 groups as structural reporters. Spectroscopically resolved carbohydrates include more than ''20 different'' small carbohydrates with more than 38 isomeric forms in addition to cereal polysaccharide fragments with suspected organoleptic and prebiotic function. Structural motifs at the cleavage sites of starch, ß-glucan and arabinoxylan fragments were identified, showing different extent and specificity of enzymatic polysaccharide cleavage during the production of different beer samples. Diffusion ordered spectroscopy supplied independent size information for the characterisation and identification of polysaccharide fragments, indicating the presence especially of high molecular weight arabinoxylan fragments in the final beer.