Impact of annealing on the hierarchical structure and physicochemical properties of waxy starches of different botanical origins.

The effect of annealing on structure and physicochemical properties of four different waxy starches were investigated to understand the mechanism of annealing in the absence of amylose. Granule morphology, X-ray pattern, molecular order, and gelatinization enthalpy remained unchanged upon annealing. Relative crystallinity of waxy corn starch increased from 42.4 to 46.1% on annealing, while it remained unchanged in other starches. Annealing increased the gelatinization temperatures, while it decreased the temperature range. Waxy potato starch showed the highest increase in To, where it was 8.1 °C after 72 h of annealing. Viscosity profiles were only slightly affected by the annealing treatment. Susceptibility towards amylolysis increased in waxy corn and waxy barley, while it decreased in waxy potato and waxy rice. This study demonstrated that even in the absence of amylose, annealing caused changes in the structure and properties, however, the extents of these changes varied depending on the botanical origin.

Characterization and differentiation of isomeric self-complementary DNA oligomers by electrospray tandem mass spectrometry.

Two series of synthetic self-complementary isomeric DNA hexamers, namely d(CAGCTG), d(CGATCG) and d(CGTACG) (M(r) 1791) and d(CATATG), d(TGATCA) and d(TGTACA) (M(r) 1790) were analysed by negative electrospray mass spectrometry. As expected, these DNA hexamers exhibited identical series of multi-charged deprotonated molecular ions. Low-energy collision-induced dissociation tandem mass spectrometric analysis of the multi-charged oligonucleotide anions [M-3H]3- and [M-4H]4- provided characteristic and distinct finger-print patterns which permitted discrimination amongst the individual isomeric DNA hexamers and allowed complete direct sequence determination.

Electrospray tandem mass spectrometry of a novel series of amphipathic functionalized ether-linked di- and trisaccharides and cyclic oligosaccharides.

Electrospray mass spectrometry (ESMS) has aided the structural characterization of a novel series of amphipathic functionalized ether-linked di- and trisaccharides, composed of units of alkyl derivatives of glucofuranose and either units of glucofuranose or diacetylgalactose. The structural elucidation of a novel eight-membered macrocyclic ether-linked disaccharide and an 11-membered macrocyclic ether-linked trisaccharide was also effected using ESMS. Low-energy collision-induced dissociation MS/MS analysis of the [M + H]+ precursor ions confirmed the characteristic fingerprint patterns obtained in the conventional electrospray spectra and proved to be a specific and very sensitive method for the detection and characterization of these novel amphipathic molecules.

Hydrolytic release, and identification by g.l.c.-m.s., of 3-deoxy-D-manno-2-octulosonic acid in the lipopolysaccharides isolated from bacteria of the Vibrionaceae.

The identification of the peracetylated methyl glycosides of 3-deoxy-D-manno-2-octulosonic acid (KDO) methyl esters was achieved by g.l.c.-m.s. These peracetylated methyl glycoside methyl esters were obtained from fully acetylated lipopolysaccharides and core oligosaccharides of representative strains of the Vibrionaceae family by the following sequence of mild reactions: acetolysis, methanolysis, and acetylation. KDO was shown to be present in all of the lipopolysaccharides (LPS), a result in direct contrast to the generally accepted view of the absence of this compound in LPS from this family of bacteria.

A new synthesis of 6-O-acylsucroses and of mixed 6,6'-di-O-acylsucroses.

Various 6-O-acylsucroses were synthesized in good yields from unprotected sucrose in N,N-dimethylformamide and the appropriate 3-acylthiazolidine-2-thiones 6 or 3-acyl-5-methyl-1,3,4-thiadiazole-2(3H)-thiones 7. A selective ionization of the free sugar by sodium hydride or triethylamine, followed by acylation with 6, gave 2-O-acylsucroses which were subjected in situ to intramolecular isomerizations using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or an aqueous solution of triethylamine to yield 6-O-acylsucroses. The later were otherwise obtained directly when sucrose was acylated with 6 or 7 in the presence of DBU. Moreover, mixed 6,6'-di-O-acylsucroses were readily obtained from 6'-monoacylates by using a Mitsunobu reaction without involving the concomitant formation of the 3',4'-epoxide.

Antifreeze glycoproteins: relationship between molecular weight, thermal hysteresis and the inhibition of leakage from liposomes during thermotropic phase transition.

Antifreeze glycoproteins (AFGP) were isolated and purified from the blood plasma of rock cod (Gadus ogac), using DEAE-Bio-gel ion exchange chromatography, followed by high performance liquid chromatography (HPLC). The purified proteins were analyzed using polyacrylamide gel electrophoresis (PAGE), and electrospray mass spectrometry. The results indicated that rock cod synthesize seven size classes of glycoproteins, ranging from 2.6 to 24 kDa, with each size class containing multiple isoforms. Antifreeze activity, as determined by thermal hysteresis, indicated that the AFGP could be separated into two groups, with the larger size classes (molecular mass>13 kDa) having approximately 3-4 times the activity of the smaller, proline containing, size classes (molecular mass<10 kDa). All of the AFGP size classes prevented leakage from dielaidoylphosphatidylcholine (DEPC) liposomes as they were cooled through their phase transition temperature, with the larger size classes being approximately 4 times as effective as the smaller ones. It is hypothesized that AFGP prevent liposomes from leaking as they pass through the phase transition temperature by binding to the phospholipid membrane.

Levels, persistence and bioavailability of organic contaminants present in marine harbor sediments impacted by raw sewage.

As part of a program to investigate the levels, fate and bioaccumulation of organic contaminants in a polluted marine harbor, organochlorine compounds including the polychlorinated biphenyls (PCBs), dichloro diphenyltrichloroethane and metabolites (DDTs), chlordanes, dibenzo-p-dioxins (Ds) and dibenzofurans (Fs), polycyclic aromatic hydrocarbons (PAHs) and sulphur hetero cycles were analysed in sediments spiked with St. John's Harbor sludge. Winter flounder (Pseudopleuronectes americanus) were exposed to these sediments containing six levels of harbor sludge during four winter months. Following exposure, sediments were reanalysed to investigate the persistence of the contaminants. The primary contaminants detected were PAHs (approximately 5000 ng/g), predominantly alkylated phenanthrenes, fluoranthene and pyrene; PCBs with a predominance of Aroclor 1260 signature (approximately 64 ng/g), DDTs (approximately 5 ng/g), hepta and octachloro dioxins and furans (approximately 0.5 ng/g) with very low levels of less chlorinated congeners. The PAHs degraded with an estimated half life of 2 to 3 months, while no significant degradation could be attributed to the other compounds. Bioaccumulation to muscle showed the potential uptake of contaminants in biota inhabiting the harbor. A dose-response relationship was observed between spiked sediments and the concentrations of PCB congener 153 and unresolved congeners 138/163/164 in muscle. Of the dioxins and furans, only 2, 3, 7, 8-tetrachlorofuran was detectable in muscle and at a similar concentration in all exposures indicating a similar bioavailability independent of the fraction of sludge in sediments. Of the PAHs, only naphthalene and methyl naphthalenes were detected in muscle, but did not display a dose-response. One bile metabolite of pyrene was quantified and demonstrates metabolism. Biota-sediment accumulation factors were of 0.2 to 7 indicating that equilibrium was approached, with the exception of the hepta and octachloro dioxins and furans which were not significantly bioaccumulated.

Complement membrane attack is required for endplate damage and clinical disease in passive experimental myasthenia gravis in Lewis rats.

Myasthenia gravis (MG) is a debilitating and potentially fatal neuromuscular disease characterized by the generation of autoantibodies reactive with nicotinic acetylcholine receptors (AChR) that cause loss of AChR from the neuromuscular endplate with resultant failure of neuromuscular transmission. A role for complement (C) in the pathology of human MG has been suggested based upon identification of C activation products in plasma and deposited at the endplate in MG. In the rat model, experimental autoimmune MG (EAMG), C depletion or inhibition restricts clinical disease, further implicating C in pathology. The mechanisms by which C activation drives pathology in MG and EAMG are unclear. Here we provide further evidence implicating C and specifically the membrane attack complex (MAC) in the Lewis rat passive EAMG model of MG. Rats deficient in C6, an essential component of the MAC, were resistant to disease induction and endplate destruction was reduced markedly compared to C6-sufficient controls. After reconstitution with C6, disease severity and endplate destruction in the C6-deficient rats was equivalent to that in controls. The data confirm the essential role of the MAC in the destruction of the endplate in EAMG and raise the prospect of specific MAC inhibition as an alternative therapy in MG patients resistant to conventional treatments.

The membrane attack pathway of complement drives pathology in passively induced experimental autoimmune myasthenia gravis in mice.

The human neuromuscular disease myasthenia gravis (MG) is characterized by the generation of autoantibodies reactive with nicotinic acetylcholine receptors (AChR) that cause loss of AChR from the neuromuscular end-plate with resultant failure of neuromuscular transmission. A role for complement (C) in AChR loss has been suggested based upon morphological identification of C at the end-plate in MG and from the effects of C inhibition in murine models. Here we provide further evidence implicating C, and specifically the membrane attack complex (MAC), in a mouse model of MG. Mice deficient in the C regulators Daf1 and/or Cd59a were tested in the model. Wild-type mice were resistant to disease while mice deficient in Daf1 had mild disease symptoms with evidence of C activation and AChR loss at end-plates. Cd59a-deficient mice had very mild disease with some muscle inflammation and essentially undamaged end-plates. In contrast, mice deficient in both C regulators developed a severe paralytic disease with marked muscle inflammation and loss of end-plates. Inhibition of MAC assembly abrogated clinical disease in these double-deficient mice, demonstrating conclusively that MAC formation was driving pathology in the model. These findings provoke us to suggest that current anti-C therapeutics targeting MAC assembly will be beneficial in MG patients resistant to conventional therapies.

Chromium trioxide oxidation of acetylated 3-amino-3,6-dideoxy-L-hexosides.

Glycosides of the fully acetylated 3-amino-3,6-dideoxy-L-hexosides in the gluco, galacto, manno, and talo configurations have been treated with chromium trioxide in acetic acid. The alpha-L-methyl glycosides, which exist in the more stable chair conformation 1C4, with an axially oriented aglycon, are resistant to this oxidation, whereas the beta-L-linked glycoside having the gluco configuration (1C4 conformation) and the alpha-L-methyl glycoside having the talo configuration (4C1 conformation), both with equatorially oriented aglycons, are oxidized to the corresponding 5-adulosonates.

Structural elucidation of the O-specific polysaccharide of the phenol-phase soluble lipopolysaccharide of Vibrio anguillarum.

The structure of the O-specific polysaccharide of the phenol-soluble cellular lipopolysaccharide of Vibrio anguillarum has been investigated. The studies involved the use of methylation analysis, partial hydrolysis with 48% hydrogen fluoride, Smith degradation, oxidation with chromium trioxide, and comprehensive proton and carbon-13 nuclear magnetic resonance studies, in which one- and two-dimensional experiments were carried out. As a result of these studies it is proposed that the O-specific polysaccharide of Vibrio anguillarum is composed of a regular heteropolymer, i.e., a main chain of (1----4)-linked 3-acetamido-3,6-dideoxy-beta-L-glucose residues alternately substituted through O-2 with side chain residues of 2-acetamido-2,6-dideoxy-alpha-D-glucose, which seem to be substituted through either O-3 or O-4 with propionyl groups (R), as in the following structure. (Formula: see text)

Structure of the lipopolysaccharide core isolated from a human strain of Aeromonas hydrophila.

Lipopolysaccharide was isolated from the cell-walls of a human strain of Aeromonas hydrophila by the aqueous phenol method in 0.58% yield (based on dry weight of bacteria). The lipopolysaccharide consisted of SR-polysaccharide, core-oligosaccharide and lipid A; there was no O-specific polysaccharide. The core had the composition D-galactose, D-glucose, D-glycero-D-manno-heptose, L-glycero-D-manno-heptose and D-glucosamine in a molar ratio of 1:1:2:4:1. Glucosamine was linked to an L-glycero-D-manno-heptose residue by a bond which was resistant to hydrolysis. The D-glucosamine-(1----7)-LD-heptose disaccharide was isolated and identified by the mass spectrum of its methylated alditol and the heptose residue not observed under normal hydrolysis conditions was easily determined after deamination of the complete core. Methylation analysis, chemical degradation, periodate and chromium trioxide oxidations and nuclear magnetic resonance (13C and 1H NMR) spectroscopy were used to identify the structure of the core oligosaccharide as: (formula: see text)

Probing the microheterogeneity of O-specific chains from Yersinia ruckeri using capillary zone electrophoresis/electrospray mass spectrometry.

The analysis of underivatized oligosaccharides arising from mild acid hydrolysis of endotoxins from Yersinia ruckeri serotype O2 was achieved using on-line capillary zone electrophoresis-electrospray mass spectrometry (CZE-ESMS). This technique provided unparalleled resolution of the different glycans obtained from purified fractions of the native endotoxins or from hydrolysis of lipopolysaccharides from Y. ruckeri. Electrophoretic conditions enabling the separation of anionic and cationic analytes were developed to determine possible sites of heterogeneity on either the core or the O-chain glycans. Structural characterization of underivatized oligosaccharides identified in the ion electropherograms was achieved using tandem mass spectrometry under low-collision energy conditions.

Synthesis of glycoconjugates derived from various lipopolysaccharides of the Vibrionaceae family.

Conjugation of simple ketoses (such as 3-deoxy-D-manno-2-octulosonic acid and N-acetylneuraminic acid) and of various O-specific polysaccharides (from Aeromonas hydrophila and Aeromonas salmonicida) to the bifunctional spacer 1,6-hexanediamine, was achieved by reductive amination. The saccharide--1-(6-amino)-hexane alkyamines obtained were converted into the corresponding isothiocyanate derivatives and coupled to the free epsilon-amino group of lysine residues of the protein carrier bovine serum albumin. In similar manner, the aldehyde group introduced by selective periodate oxidation into the partially O-deacylated lipopolysaccharide of Vibrio anguillarum was conjugated to 1,6-hexanediamine, converted into the corresponding isothiocyanate and covalently attached to bovine serum albumin.