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The tropical tree Calophyllum brasiliense Cambess. (Clusiaceae) grows in rain forests from Brazil to Mexico. Its leaves, as well as those of other Calophyllum species, are rich sources of chromanone acids, such as apetalic acid, isoapetalic acid, and their derivatives. Apetalic acid has shown significant antimycobacterial activity. The biological activity of apetalic acid has been related to the configuration of three asymmetric centers and the stereochemistry of the molecule; however, the C-19 configuration in the acidic side chain has not been fully resolved. For this reason, the unequivocal determination of the absolute configuration by means of X-ray crystallography in a sample of unique homogeneous apetalic acid stereoisomer was the most important point to start this study. Chiral amides were prepared using the carboxyl group. We determined the C-19 stereochemistry of apetalic acid and its specific chiral derivatives using NMR, X-ray diffraction, and molecular mechanics. Finally, we observed that steric hindrance in the side chain of apetalic acid leads to restriction of rotation around the pivotal linkage C-10–C-19, establishing chiral centers at C2(R), C3(S), and C19(R). We were able to separate the derivatives of these two high-rotatory-barrier conformers of apetalic acid by forming diastereoisomeric amides with phenylglycine methyl ester having a chiral center at C-2′. Our results confirmed the existence of atropisomerism in the apetalic acid molecule.

The capsular polysaccharide (CPS) of Streptococcus suis defines various serotypes based on its composition and structure. Though serotype switching has been suggested to occur between S. suis strains, its impact on pathogenicity and virulence remains unknown. Herein, we experimentally generated S. suis serotype-switched mutants from a serotype 2 strain that express the serotype 3, 4, 7, 8, 9, or 14 CPS. The effects of serotype switching were then investigated with regards to classical properties conferred by presence of the serotype 2 CPS, including adhesion to/invasion of epithelial cells, resistance to phagocytosis by macrophages, killing by whole blood, dendritic cell-derived pro-inflammatory mediator production and virulence using mouse and porcine infection models. Results demonstrated that these properties on host cell interactions were differentially modulated depending on the switched serotypes, although some different mutations other than loci of CPS-related genes were found in each the serotype-switched mutant. Among the serotype-switched mutants, the mutant expressing the serotype 8 CPS was hyper-virulent, whereas mutants expressing the serotype 3 or 4 CPSs had reduced virulence. By contrast, switching to serotype 7, 9, or 14 CPSs had little to no effect. These findings suggest that serotype switching can drastically alter S. suis virulence and host cell interactions.

Streptococcus suis serotype 2 strains can cause severe infections in both swine and humans. The capsular polysaccharide (CPS) of S. suis defines various serotypes based on its composition and structure. Though serotype switching from serotype 2 has been suggested to occur between S. suis strains, its impact on pathogenicity and virulence remains unknown. Herein, we experimentally generated S. suis serotype-switched mutants from a serotype 2 strain (SS2) that express the serotype 3, 4, 7, 8, 9, or 14 CPS (SS2to3, SS2to4, SS2to7, SS2to8, SS2to9, and SS2to14, respectively). The effects of serotype switching were then investigated with regards to classical properties conferred by presence of the serotype 2 CPS, including adhesion to/invasion of porcine tracheal epithelial cells, resistance to phagocytosis by murine macrophages, killing by murine and porcine whole blood, and dendritic cell-derived pro-inflammatory mediator production. Results demonstrated that these properties on host cell interactions were differentially modulated depending on the switched serotypes. Using a mouse model of systemic infection, SS2to8 was demonstrated to be hyper-virulent, with animals rapidly succumbing to septic shock, whereas SS2to3 and SS2to4 were less virulent than SS2 because of a reduced systemic inflammatory host response. By contrast, switching to serotype 7, 9, or 14 CPSs had little to no effect. Finally, development of clinical signs in a porcine model of infection was only observed following infection with SS2, SS2to7, and SS2to8. Taken together, these findings suggest that serotype switching can differentially modulate S. suis host cell interactions and virulence depending on the CPS type expressed.ImportanceStreptococcus suis serotype 2 is the most frequently type associated with swine and zoonotic infections. While the serotype 2 CPS is required for virulence and pathogenesis, little information is available regarding that of other serotypes and how differences in serotype can directly affect host cell interactions and virulence. Herein, we constructed serotype-switched mutants from a serotype 2 strain and demonstrated that serotype switching can shift and modulate the S. suis host cell interactions and virulence in vivo. Among the serotype-switched mutants, the mutant expressing the serotype 8 CPS, whose composition and structure are identical to that of the human pathogen Streptococcus pneumoniae serotype 19F, was hyper-virulent, whereas mutants expressing the serotype 3 or 4 CPSs had reduced virulence. These results demonstrate that serotype switching can drastically alter S. suis phenotype. Consequently, further importance and attention should be given to the phenomenon of serotype switching and the possible emergence of hyper-virulent isolates.

Streptococcus suis is an encapsulated bacterium and one of the most important swine pathogens and a zoonotic agent for which no effective vaccine exists. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. In addition to the previously known serotypes 2 and 14, which are nonimmunogenic, we have recently purified and described the CPS structures for serotypes 1, 1/2, 3, 7, 8, and 9. Here, we aimed to elucidate how these new structurally diverse CPSs interact with the immune system to generate anti-CPS antibody responses. CPS-stimulated dendritic cells produced significant levels of C–C motif chemokine ligand 3 (CCL3), partially via Toll-like receptor 2 (TLR2)- and myeloid differentiation factor 88-dependent pathways, and CCL2, via TLR-independent mechanisms. Mice immunized with purified serotype 3 CPS adjuvanted with TiterMax Gold produced an opsonizing IgG response, whereas other CPSs or adjuvants were negative. Mice hyperimmunized with heat-killed S. suis serotypes 3 and 9 both produced anti-CPS type 1 IgGs, whereas serotypes 7 and 8 remained negative. Also, mice infected with sublethal doses of S. suis serotype 3 produced primary anti-CPS IgM and IgG responses, of which only IgM were boosted after a secondary infection. In contrast, mice sublethally infected with S. suis serotype 9 produced weak anti-CPS IgM and IgG responses following a secondary infection. This study provides important information on the divergent evolution of CPS serotypes with highly different structural and/or biochemical properties within S. suis and their interaction with the immune system.

The EPS Database (EPS-DB) is a web-based, platform-independent database of bacterial exopolysaccharides (EPSs) providing access to detailed structural, taxonomic, growth conditions, functional properties, genetic, and bibliographic information for EPSs. It is freely available on the Internet as a website at http://www.epsdatabase.com. Several structural data representation schemes are used following the most commonly accepted formats. This guarantees full interoperability with other structural, experimental, and functional databases in the area of glycoscience. The scientific usage of EPS-DB throughout a user-friendly interface is presented with a subsection of the database exemplified by EPSs from lactic acid bacteria.

Interactions of exopolysaccharides and proteins are of great importance in food science, but complicated to analyze and quantify at the molecular level. A surface plasmon resonance procedure was established to characterize binding of seven structure-determined, branched hetero-exopolysaccharides (HePSs) of 0.14–4.9 MDa from lactic acid bacteria to different milk proteins (β-casein, κ-casein, native and heat-treated β-lactoglobulin) at pH 4.0–5.0. Maximum binding capacity (RUmax) and apparent affinity (KA,app) were HePS- and protein-dependent and varied for example 10- and 600-fold, respectively, in the complexation with native β-lactoglobulin at pH 4.0. Highest RUmax and KA,app were obtained with heat-treated β-lactoglobulin and β-casein, respectively. Overall, RUmax and KA,app decreased 6- and 20-fold, respectively, with increasing pH from 4.0 to 5.0. KA,app was influenced by ionic strength and temperature, indicating that polar interactions stabilize HePS–protein complexes. HePS size as well as oligosaccharide repeat structure, conferring chain flexibility and hydrogen bonding potential, influence the KA,app.

Despite a very large number of bacterial exopolysaccharides have been reported, detailed knowledge on their molecular structures and associative interactions with proteins is lacking. Small-angle X-ray scattering, dynamic light scattering and analytical ultracentrifugation (AUC) were used to characterize the interactions of six lactic acid bacterial heteroexopolysaccharides (HePS-1–HePS-6) with β-lactoglobulin (BLG). Compared to free HePSs, a large increase in the X-ray radius of gyration RG, maximum length L and hydrodynamic diameter dH of HePS-1–HePS-4 mixed with BLG revealed strong aggregation, the extent of which depended on the compact conformation and degree of branching of these HePSs. No significant effects were observed with HePS-5 and HePS-6. Turbidity and AUC analyses showed that both soluble and insoluble BLG–HePS complexes were formed. The findings provide new insights into the role of molecular structures in associative interactions between HePSs and BLG which has relevance for various industrial applications.

The impact of fatty acids on the growth of lactobacilli and bifidobacteria was studied to gain insight into the beneficial effects of conjugated linoleic acid (CLA) and probiotics. Milk was enriched with CLA by adding 4% safflower oil to the cow diet, by emulsifying synthetic CLA as free fatty acids, or by emulsifying CLA as triacylglycerol, then adjusted to 1.0% or 3.25% fat and digested in a gastro-intestinal simulator. The residual fatty acid contents of milk samples containing initial fat contents of 1.0% and 3.25% were significantly different after 6 h digestion. Milk enriched with synthetic CLA and adjusted to 1.0% fat appeared to provide a digest that allowed growth of both Lactobacillus rhamnosus R0011 and Lb. rhamnosus LGG; adjustment to 3.25% fat decreased viable LGG slightly. Digestion of milk containing 3.25% fat appeared to yield a residual fatty acid mixture that may be slightly bactericidal or simply bacteriostatic.

The intestinal absorption of fatty acids from milk, especially conjugated linoleic acids (CLA), was evaluated using an in vitro simulator. Milk was enriched with c9,t11 CLA by feeding a cow a diet containing 4% safflower oil or by emulsifying synthetic CLA in the form of free fatty acid or triacylglycerol, then standardized to 1.0% or 3.25% fat. Fatty acid bioavailability depended on chain length, presence of double bonds, and the percentage of fat. Absorption was more efficient in the presence of 1.0% fat. The bioavailability of c9,t11 CLA at 3.25% fat was 89% in the milk from the cow fed the safflower-oil diet, and 85% and 71% in milk enriched with synthetic free CLA and synthetic CLA in the form of triacylglycerol, respectively. At 1.0% fat, these values were 90%, 99% and 96%, respectively, suggesting that bioavailability of CLA in milk is affected by the percentage of fat present.

Streptococcus agalactiae (also known as group B Streptococcus [GBS]) and Streptococcus suis are encapsulated streptococci causing severe septicemia and meningitis. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. The mechanisms underlying anti-CPS antibody responses are not fully elucidated, but the biochemistry of CPSs, particularly the presence of sialic acid, may have an immunosuppressive effect. We investigated the ability of highly purified S. suis and GBS native (sialylated) CPSs to activate dendritic cells (DCs), which are crucial actors in the initiation of humoral immunity. The influence of CPS biochemistry was studied using CPSs extracted from different serotypes within these two streptococcal species, as well as desialylated CPSs. No interleukin-1β (IL-1β), IL-6, IL-12p70, tumor necrosis factor alpha (TNF-α), or IL-10 production was observed in S. suis or GBS CPS-stimulated DCs. Moreover, these CPSs exerted immunosuppressive effects on DC activation, as a diminution of gamma interferon (IFN-γ)-induced B cell-activating factor of the tumor necrosis factor family (BAFF) expression was observed in CPS-pretreated cells. However, S. suis and GBS CPSs induced significant production of CCL3, via partially Toll-like receptor 2 (TLR2)- and myeloid differentiation factor 88 (MyD88)-dependent pathways, and CCL2, via TLR-independent mechanisms. No major influence of CPS biochemistry was observed on the capacity to induce chemokine production by DCs, indicating that DCs respond to these CPSs in a patterned way rather than a structure-dedicated manner.

Knowledge on molecular structure of exopolysaccharides (EPSs) and their roles in the associative interactions with proteins is essential to understand the relationship between their structure, physical and rheological properties. Despite their importance, no detailed molecular characterization of bacterial EPSs and their associative interactions with proteins have been reported up to now. By combining X-ray solutionscattering (SAXS), dynamic light scattering (DLS) and analytical ultracentrifugation (AUC) in conjunction with scattering modeling, we have characterized four different heteroexopolysaccharides (HePS-1–HePS-4) from lactic acid bacteria (LAB) and their interactions with β-lactoglobulin. We have previously shown that these HePSs exhibited a compact conformation in solution. Here, SAXS data for HePSs (HePS-1–HePS-4) complexes with β-lactoglobulin showed that β-lactoglobulin aggregated strongly with these HePSs. β-lactoglobulin exists as a dimer at pH 4 in the absence of HePSs. When mixed with HePSs, SAXS analysis showed that β-lactoglobulin formed large aggregates. DLS also showed formation of large aggregates of β-lactoglobulin with HePSs, thus validating SAXS data. Turbidity and AUC data indicated that both soluble and insoluble BLG–HePSs complexes were formed. This study provides new insights into the role of molecular structures in associative interactions between HePSs and BLG which has relevance for various industrial applications.

Molecular structures of exopolysaccharides are required to understand their functions and the relationships between the structure and physical and rheological properties. Small-angle X-ray scattering and dynamic light scattering were used in conjunction with molecular modeling to characterize solution structures of three lactic acid bacterial heteroexopolysaccharides (HePS-1, HePS-2, and HePS-3). Values of radius of gyration RG, cross-sectional radius of gyration Rxs, approximate length L, and hydrodynamic diameter were not directly proportional to the molar mass and indicated the HePSs adopted a compact coil-like rather than an extended conformation. Constrained molecular modeling of 15000 randomized HePS-1 conformers resulted in five best-fit structures with R factor of 3.9-4.6% revealing random coil-like structure. φ and ψ angle analysis of glycosidic linkages in HePS-1 structures suggests Galf residues significantly influence the conformation. Ab initio scattering modeling of HePS-2 and HePS-3 gave excellent curve fittings with χ2 of 0.43 and 0.34 for best-fit models, respectively, compatible with coil-like conformation. The findings disclose solution behavior of HePS relevant for their interactions with biomacromolecules, for example, milk proteins.

The present study evaluated the in vitro bioaccessibility of milk fatty acids (FA) and cis-9, trans-11 18:2 using the TIM-1 gastrointestinal model from TNO, Zeist, The Netherlands. Milk naturally enriched with cis-9, trans-11 18:2 (46 mg g−1 FA) was obtained from a lactating dairy cow fed safflower oil. The other treatments consisted of synthetic conjugated linoleic acids as triacylglycerols or free FA incorporated into milk containing a low level of cis-9, trans-11 18:2. Treatments were standardised at 3.25% fat and pasteurised. Chyme was analysed for FA composition and lipid classes. FA bioaccessibility was 81.8 ± 3.1% and was not different between treatments. The degree of absorption of saturated FA decreased with chain length. Bioaccessibility was higher for unsaturated FA than for their saturated counterparts. Bioaccessibility of cis-9, trans-11 18:2 was similar among treatments and averaged 87.0 ± 3.4%. Results suggest that regiospecific distribution of cis-9, trans-11 18:2 on triacylglycerol structure does not influence its accessibility for intestinal absorption.

The structure and stereochemistry of four sesquiterpene lactones, budlein A (1), zaluzanin A (2), and glaucolides D (3a) and E (3b), isolated from Mexican Asteraceae species, for which only partial NMR data could be found in the literature, were determined. A combination of 1D and 2D high-resolution NMR experiments, such as DEPT, COSY, NOESY, ROESY, HMQC, HSQC, and HMBC, were used to completely assign the 1H and 13C spectra. The crystal structures of zaluzanin A (2) and glaucolide E (3b) were also determined. Glaucolides D and E have been previously reported to relax KCl-induced contraction in rat uterine smooth muscle; therefore, the effects of zaluzanin A and budlein A were examined in the same model. It was found that both compounds can relax contraction induced by KCl, but only zaluzanin A induced relaxation when contraction was induced with oxytocin. The preliminary biological test results according to these profiles are reported in this paper.Key words: NMR data, X-ray data, sesquiterpene lactones, Mexican Asteraceae, smooth muscle relaxant.

The capsular polysaccharide (CPS) is a major virulence factor in many encapsulated pathogens, as it is the case for Streptococcus suis, an important swine pathogen and emerging zoonotic agent. Moreover, the CPS is the antigen at the origin of S. suis classification into serotypes. Hence, analyses of the CPS structure are an essential step to dissect its role in virulence and the serological relations between important serotypes. Here, the CPSs of serotypes 1 and 1/2 were purified and characterized for the first time. Chemical and spectroscopic data gave the following repeating unit sequences: [6)[Neu5Ac(α2-6)GalNAc(β1-4)GlcNAc(β1-3)]Gal(β1-3)Gal(β1-4)Glc(β1-]n (serotype 1) and [4)[Neu5Ac(α2-6)GalNAc(β1-4)GlcNAc(β1-3)]Gal(β1-4)[Gal(α1-3)]Rha(β1-4)Glc(β1-]n (serotype 1/2). The Sambucus nigra lectin, which recognizes the Neu5Ac(α2-6)Gal/GalNAc sequence, showed binding to both CPSs. Compared with previously characterized serotype 14 and 2 CPSs, N-acetylgalactosamine replaces galactose as the sugar bearing the sialic acid residue in the side chain. Serological analyses of the cross-reaction of serotype 1/2 with serotypes 1 and 2 and that between serotypes 1 and 14 suggested that the side chain, and more particularly the terminal sialic acid, constitutes one important epitope for serotypes 1/2 and 2. The side chain is also an important serological determinant for serotype 1, yet sialic acid seems to play a limited role. In contrast, the side chain does not seem to be part of a major epitope for serotype 14. These results contribute to the understanding of the relationship between S. suis serotypes and provide the basis for improving diagnostic tools.

The neutral exopolysaccharide (NPS) of Lactobacillus delbrueckii subsp. bulgaricus strain OLL1073R-1 was purified and characterized. The molecular mass was 5.0×10 6 g/mol. Sugar and absolute configuration analyses gave the following composition: d -Glc, 1; d -Gal, 1.5. The NPS was also submitted to periodate oxidation followed by borohydride reduction and Smith degradation. Sugar and methylation analyses, 1 H and 13 C nuclear magnetic resonance, and mass spectrometry of the NPS or of its specifically modified products allowed determining the repeating unit sequence: {2)Glc(α1–3)Glc(β1–3)[Gal(β1–4)]Gal(β1–4)Gal(α1–} n . The structure is compared to that of exopolysaccharides produced by other Lactobacillus bulgaricus strains.

Exopolysaccharides (EPSs) were isolated and purified from Lactobacillusrhamnosus strains RW-9595M, which has been shown to possess cytokine-stimulating activity, and R grown under various fermentation conditions (carbon source, incubation temperature and duration). Identical 1H NMR spectra were obtained in all cases. Molecular masses were determined by gel permeation chromatography. The primary structure was elucidated using chemical and spectroscopic techniques. Organic acid, monosaccharide and absolute configuration analyses gave the following composition: pyruvate, 1; d-glucose, 2; d-galactose, 1; and l-rhamnose, 4. Methylation analysis indicated the presence of three residues of 3-linked rhamnose, and one residue each of 2,3-linked rhamnose, 2-linked glucose, 3-linked glucose and 4,6-linked galactose. The EPS was submitted to periodate oxidation followed by borohydride reduction. Monosaccharide analysis of the resulting polysaccharide gave the new composition: rhamnose, 4; and glucose, 1. Methylation analysis confirmed the loss of the 2-linked glucose and 4,6-linked galactose residues. On the basis of one- and two-dimensional 1H and 13C NMR data, the structure of the native EPS was consistent with the following heptasaccharide repeating unit: {3Rhaα-3Glcβ-3[Gal4,6(R)Pyα-2]Rhaα-3Rhaα-3Rhaα-2Glcα-}n where Rha corresponds to rhamnose (6-deoxymannose) and Py corresponds to pyruvate acetal. Complete 1H and 13C assignments are reported for the native and the corresponding pyruvate-hydrolysed polysaccharide. Electrospray MS and MS/MS data are given for the oligosaccharide produced by Smith degradation.

Productions de TNF, IL-6, IL-12 et IFN-γ chez des cellules immunocompetentes traitees avec des exopolysaccharides du Lactobacillus rhamnosus RW-9595M. Difference entre les reponses de cellules de sang peripherique humain et de splenocytes de souris. Les exopolysaccharides (EPS) du Lactobacillus rhamnosus RW- 9595M ont ete prepares a partir de cultures bacteriennes, isoles, concentres, fractionnes et testes in vitro quant a leur potentiel immunomodulateur sur des splenocytes de souris C57B1/6 et BALB/c, sur la lignee macrophagique murine RAW.264.7 et sur des cellules mononuclees du sang humain provenant de 14 donneurs. Nous avons fractionne ces EPS en fonction des tailles moleculaires, avec des membranes filtrantes de differentes porosites. Les fractions correspondent aux tailles suivantes: F1: > 1000 kg.mol -1 , F2: 1000-100 kg.mol -1 , F3: 100-10 kg.mol -1 et F4: < 10 kg.mol -1 . Les EPS concentres, non fractionnes, ainsi que F1, se sont averes legerement mitogenes sur des splenocytes de la souris, ou des cellules sanguines humaines, cultivees 2-3 jours. Dans le dernier cas, F3 manifeste egalement cette propriete. Les EPS non fractionnes, ainsi que F1, sont capables de provoquer la production de TNF, IL-6, IL-12p40 apres 6 ou 24 h de culture, l'origine des cellules etant importante pour l'intensite de la reponse. Apres 24, 48 et 72 h de culture, les EPS non fractionnes, ainsi que F1, ont provoque la production d'interferon gamma par des splenocytes de souris C57B1/6 et BALB/c. Au contraire, dans le cas des cellules sanguines humaines, aucune production d'interferon gamma n'a pu etre observee, en depit d'une bonne reponse en IL-12p40. Ces resultats suggerent que les EPS de bacteries lactiques peuvent stimuler certaines fonctions immunitaires chez des sujets aptes a repondre.

Crocetin derivatives were extracted and purified from saffron stigmas and gardenia fruits by reversed-phase HPLC. They were characterized by 1D and 2D 1H and 13C NMR and UV−vis spectroscopies and mass spectrometry. The following compounds were found in saffron: all-trans-crocetin di(β-d-gentiobiosyl) ester 1a, all-trans-crocetin β-d-gentiobiosyl-β-d-glucosyl ester 1b, all-trans-crocetin di(β-d-glucosyl) ester 1c, and all-trans-crocetin mono(β-d-gentiobiosyl) ester 1d, as well as 13-cis-crocetin di(β-d-gentiobiosyl) ester 2a and 13-cis-crocetin β-d-gentiobiosyl-β-d-glucosyl ester 2b. Compounds 1a, 1b, and 1d were also present in gardenia, in addition to all-trans-crocetin mono(β-d-glucosyl) ester 1e. Keywords: Saffron; gardenia; Crocus sativus; Gardenia jasminoides; crocetin derivatives; carotenoid pigments; mass spectrometry; UV−vis spectroscopy; NMR spectroscopy

The capsular polysaccharide (CPS) of Streptococcus suis serotype 14 was purified, chemically modified, and characterized. Sugar and absolute configuration analyses gave the following CPS composition: d-Gal, 3; d-Glc, 1; d-GlcNAc, 1; d-Neu5Ac, 1. The Sambucus nigra lectin, which recognizes the Neu5Ac(α2–6)Gal/GalNAc sequence, showed binding to the native CPS. Sialic acid was found to be terminal, and the CPS was quantitatively desialylated by mild acid hydrolysis. It was also submitted to periodate oxidation followed by borohydride reduction and Smith degradation. Sugar and methylation analyses,1H and13C nuclear magnetic resonance, and mass spectrometry of the native CPS or of its specifically modified products allowed to determine the repeating unit sequence: [6)[Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)]Gal(β1–3)Gal(β1–4)Glc(β1–]n. S. suis serotype 14 CPS has an identical sialic acid-containing side chain as serotype 2 CPS, but differs by the absence of rhamnose in its composition. The same side chain is also present in group B Streptococcus type Ia CPS, except that in the latter sialic acid is 2,3- rather than 2,6-linked to the following galactose. A correlation between the S. suis CPS sequence and genes of the serotype 14 cps locus encoding putative glycosyltransferases and polymerase responsible for the biosynthesis of the repeating unit is proposed.

Streptococcus suis type 2 is a major swine pathogen and a zoonotic agent, causing meningitis in both swine and humans. S. suis infects the host through the respiratory route, reaches the bloodstream, and persists until breaching into the central nervous system. The capsular polysaccharide (CPS) of S. suis type 2 is considered a key virulence factor of the bacteria. Though CPS allows S. suis to adhere to the membrane of cells of the immune system, it provides protection against phagocytosis. In fact, nonencapsulated mutants are easily internalized and killed by macrophages and dendritic cells. The objective of this work was to study the molecular mechanisms by which the CPS of S. suis prevents phagocytosis. By using latex beads covalently linked with purified CPS, it was shown that CPS itself was sufficient to inhibit entry of both latex beads and bystander fluorescent beads into macrophages. Upon contact with macrophages, encapsulated S. suis was shown to destabilize lipid microdomains at the cell surface, to block nitric oxide (NO) production during infection, and to prevent lactosylceramide accumulation at the phagocytic cup during infection. In contrast, the nonencapsulated mutant was easily internalized via lipid rafts, in a filipin-sensitive manner, leading to lactosylceramide recruitment and strong NO production. This is the first report to identify a role for CPS in lipid microdomain stability and to recognize an interaction between S. suis and lactosylceramide in phagocytes.

The capsular polysaccharide is a critical virulence factor of the swine and zoonotic pathogen Streptococcus suis serotype 2. The capsule of this bacterium is composed of five different sugars, including terminal sialic acid. To evaluate the role of sialic acid in the pathogenesis of the infection, the neuC gene, encoding for an enzyme essential for sialic acid biosynthesis, was inactivated in a highly virulent S. suis serotype 2 strain. Using transmission electron microscopy, it was shown that inactivation of neuC resulted in loss of expression of the whole capsule. Compared to the parent strain, the ΔneuC mutant strain was more phagocytosed by macrophages and was also severely impaired in virulence in a mouse infection model. Both native and desialylated S. suis serotype 2 purified capsular polysaccharides were recognized by a polyclonal anti-whole cell S. suis serotype 2 serum and a monospecific polyclonal anti-capsule serotype 2 serum. In contrast, only the native capsular polysaccharide was recognized by a monoclonal antibody specific for the sialic acid moiety of the serotype 2 capsule. Together, our results infer that sialylation of S. suis serotype 2 may be essential for capsule expression, but that this sugar is not the main epitope of this serotype.

The reaction of dibenzalcyclohexanone with hydroxylamine hydrochloride afforded three compounds 1–3 including the aziridine 3 showing a 3′,3a′-trans configuration. Now we report on the isolation of a new aziridine 4, possessing a 3′,3a′-cis configuration. Its structure was deduced by 2D nmr and single crystal X-ray diffraction studies.

The capsular polysaccharide (CPS) of Streptococcus suis serotype 2 was isolated, purified, chemically modified, and characterized. Sugar and absolute configuration analyses of the CPS gave the following composition: d-Gal, 3; d-Glc, 1; d-GlcNAc, 1; d-Neu5Ac, 1; l-Rha, 1. Sialic acid was found to be terminal, and the CPS was quantitatively desialylated by mild acid hydrolysis. The CPS was also submitted to periodate oxidation followed by borohydride reduction and Smith degradation. Sugar and methylation analysis,1H and13C nuclear magnetic resonance, and mass spectrometry of the native CPS or of its specifically modified products allowed to determine the repeating unit sequence: [4)[Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)]Gal(β1–4)[Gal(α1–3)]Rha(β1–4)Glc(β1-]n. The backbone sequence was found to be identical to that of Streptococcus agalactiae or group B Streptococcus (GBS) type VIII and Streptococcus pneumoniae type 23F. The S. suis CPS shares the sequence Neu5Ac-Gal-GlcNAc-Gal in common with GBS types Ia, Ib, II, III, and IV CPSs but differs from them by the presence of rhamnose and the fact that sialic acid is 2,6- rather than 2,3-linked to the following Gal. A correlation between the S. suis CPS sequence and genes of the serotype 2 cps locus encoding putative enzymes responsible for the biosynthesis of the repeating unit was tentatively established.

It is known that dilute acid hydrolysis of cellulose at high temperature results in yields not exceeding 60–65% of the potential glucose. All the models presented in the literature are based on the sequence: cellulose #x00BB; glucose #x00BB; degradation products. The underlying assumption of this sequence is that the unconverted cellulose retains its chemical integrity during the course of the hydrolysis reaction. Experimental evidence based on physico-chemical characterization of cellulosic residues obtained from dilute acid hydrolysis processes allows us to propose a new mechanism for cellulose hydrolysis accounting for its degradation in the solid state. Such degradation results in the formation of a non- carbohydrate residue.

The structure and configuration of salvianduline C, a novel 5,6-secoclerodane diterpenoid isolated from Salvia lavanduloides , have been established by high resolution NMR spectroscopy and X-ray crystallographic analysis.

A new series of linear and permanently charged poly(amidoammonium) salts were synthesized in order to investigate the influence of their ionic and hydrophobic contents on both the cytotoxicity and the transfection mediated by polycation-DNA complexes. The poly(amidoammonium) salts were prepared by chemical modification of a parent poly(amidoamine) containing two tertiary amino groups per structural unit: one incorporated into the main chain and the other fixed at the end of a short bismethylene spacer. The permanent charges were introduced through a quaternization reaction involving iodomethane or 1-iodododecane as an alkylating agent. Under appropriate conditions, the methylation reaction was found to be regioselective, allowing the quaternization of either the side chains or both the side chains and the backbone. Under physiological salt conditions (150 mM NaCl), all of the poly(amidoammonium) salts self-assembled with DNA to form complexes. High proportions of highly quaternized polycation provided better defined morphology to the polycation-DNA complexes. Complexes formed from unquaternized polycation were less cytotoxic than branched poly(ethyleneimine) (25 kDa). At high polycation-DNA weight ratios, the introduction of permanent charges generated a significant increase in the cytotoxicity, but no patent correlation could be established with the amount and the position of the permanent charges. Only complexes formed from polycations with quaternized backbone were able to generate significant gene expression, which was putatively attributed to a better defined toroidal-like morphology together with a higher stability, as suggested by zeta potential measurements. The incorporation of dodecane side chains on highly charged polycations severely amplified the cytotoxicity so that, in return, the transfection level was dramatically affected.

Antimicrobial films were prepared by dissolving chitosan into hydrochloric, formic, acetic, lactic and citric acid solutions. Below 40 degrees C, the counter ions could be classified into two groups based on their effect on zero-shear-rate viscosity in 2% solutions of organic acids. Chloride and citrate produced solutions with much lower viscosities than formate, acetate and lactate. At higher temperatures, these differences vanished, and the activation energies of viscous flow were all similar between 40 and 60 degrees C. Films prepared from these solutions were evaluated in tension for Young's modulus, stress and elongation at yield and break points. Films made from hydrochloric, formic and acetic acids were hard and brittle, whereas those from lactic and citric acids were soft and could be stretched. Good correlation was found between Young's modulus and volume of the counter ion. Film properties are essentially governed by the volume of the counter ion and not by the interactions between this counter ion and the macromolecule. Results suggest that acetate has the maximum molecular volume above which the film strength decreases very rapidly.

A conformational study is performed on the acylated erythromycin and erythralosamine derivatives from comparison between experimental results (NMR) and theoretical calculations by Molecular Dynamics (MD) in attempts to correlate their conformations with their abilities to generate cytochrome P450-nitroso metabolite complexes in vitro. As the 3'-dimethyl-amino function of the desosamine is metabolized and responsible for the interaction with cytochrome P450, its position, mobility and steric hindrance in the proximity of this functional group are related to its biological properties. The major conformations of the lactone ring were termed A (A1, A2, A3) and B (B1, B2), and this macrocycle flexibility induced five different orientations a, b, c, d and e for the desosamine sugar. Conformations A and B differ in many ways but the major change is the inward folding of the C(3) fragment in B. Conformer a exhibits an orientation of the desosamine nearly perpendicular to the macrocycle whereas the two units are in the same plane in conformations c and e. For conformation b, the cladinose unit lifts up above the macrocycle. Conformation d exhibits a turned-back cladinose. In the erythromycin derivatives esterification at the beta position to the N(CH3)2 group of the desosamine reduces the degree of freedom of the macrocyclic lactone ring which corresponds to conformation A only. The desosamine sugar was found to be perpendicular to the macrocycle (a conformer) and both sugar groups are parallel to reduce the steric energy. In the erythralosamine derivatives, the macrocycle is always present as conformation B with the two conformations b and c of the sugar rings. The steric parameters favour the b conformers in which the amino group is tilted up, while in 3,2'-dibenzoylated stacking aromatic attraction stabilizes the planar c conformer. Both isomers are thus shown to adopt well-defined conformations and to be well-adapted for a comparative structure-activity correlation studies. There is a significant relationship between the conformation b and the formation of cytochrome P450-nitroso metabolite complexes.

In order to get insights into the effects of cholesterol on protein activity, the lytic power of melittin on 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC)/ cholesterol mixtures was studied using solid-state deuterium and phosphorus-31 nuclear magnetic resonance spectroscopy (2H and 31P-NMR). After incubation, melittin disrupts pure DPPC vesicles, leading to the formation of small lipid/peptide complexes below the phase transition temperature ( T m , whereas large bilayer assemblies are reformed above T m ; the transition between these two species is thermally reversible. This study reveals that cholesterol modifies this thermal behavior and that this modulation of the lytic power of melittin is indirect, since it is essentially related to the original effect of the sterol on the thermotropism of pure lipid bilayers. It is known that melittin does not lyse gel phase DPPC bilayers spontaneously. Our study shows that the addition of large amounts of sterol (30 mol%) does not promote the spontaneous lysis at 26°C, despite the increased fluidity of the lipid system. The lysis takes place around 32°C, regardless of the cholesterol concentration. This study also shows that high concentrations of cholesterol ( ≧ 30% ) in DPPC bilayer inhibit the lysis. It is proposed that the tight lipid packing due to high cholesterol concentrations prevents the penetration of melittin into the bilayer. When melittin interacts with cholesterol-rich bilayers (30 mol%), the lysis is only partial, and leads to the formation of small cholesterol-depleted particles. Finally, DPPC which bears deuteriated acyl chains was used to determine the infuence of melittin on the orientational order of the lipid chains in the large assemblies. The quadrupolar splittings obtained in the presence of melittin are not considerably different than those obtained in the absence of melittin.

Amphiphilic dimethylaminopyridinium alkyl polymethacrylates (aPPs) were tested for gene complexation, cell cytotoxicity and in vitro gene expression for use as gene delivery agents. The aminopyridinium groups neutralized by bromide or octylsulfonate counterions were terminal moieties of side-chain spacers containing 8, 12 or 16 methylene units. This investigation measured the impact of the spacer length and the chemical nature of the counterion on the physicochemical properties and biological activity of the polyplexes formed by the complexation with DNA. The aPPs self-assembled with DNA by neutralizing the DNA phosphate charges through the pyridinium moieties. The degree of DNA condensation was higher for shorter spacer (n = 8, 12) and bromide-neutralized aPPs. Several aPP–DNA complexes formed well-defined nanoparticles, which were usually, but not always, positively charged. Their sizes ranged from 30 to 150 nm and in some cases had an internal lamellar structure visible by TEM. All of the aPPs were found to be much less cytotoxic than branched poly(ethyleneimine) [(PEI), 25 kDa]. The degree of cytotoxicity of the aPPs depended mildly on their spacer length and counterion: a longer spacer (n = 16) decreased the cell viability more than shorter spacers and, at the highest aPP concentrations tested, bromide counterions more than octylsulfonate counterions. The transfection efficiency also depended on the spacer length and counterion type. Polyplexes obtained from the bromide-neutralized aPPs with the n = 12 spacer at an aPP/DNA weight ratio of 2.5, for which negatively charged nanoparticles were formed, were found to be as efficient as PEI-based polyplexes. Interestingly, this demonstrates that endosomolytic fragments and positively charged polyplex surfaces are not required for efficient gene expression.

In this study, we report that Lactococcus lactis strains producing exopolysaccharides (EPS) are sensitive to virulent phages. Eight distinct lytic phages (Q61 to Q68) specifically infecting Eps + strains were isolated in 47 buttermilk samples obtained from 13 North American factories. The eight phages were classified within the 936 species by the multiplex PCR method, indicating that these phages are not fundamentally distinct from those infecting Eps − L. lactis strains. The host range of these phages was determined with 19 Lactococcus strains, including 7 Eps + and 12 Eps − cultures. Three phages (Q62, Q63, and Q64) attacked only the Eps + strain SMQ-419, whereas the five other phages (Q61, Q65, Q66, Q67, and Q68) infected only the Eps + strain SMQ-420. The five other Eps + strains (H414, MLT2, MLT3, SMQ-461, and SMQ-575) as well as the 12 Eps − strains were insensitive to these phages. The monosaccharide composition of the polymer produced by the seven Eps + strains was determined. The EPS produced by strains MLT3, SMQ-419, and SMQ-575 contained glucose, galactose, and rhamnose. The EPS fabricated by H414 contained only galactose. The EPS made by MLT2, SMQ-420, and SMQ-461 contained glucose and galactose. These findings indicate that the sugar composition of the EPS has no effect on phage sensitivity. The plasmid encoding the eps operon was cured from the two phage-sensitive strains. The cured derivatives were still phage sensitive, which indicates that EPS are not necessary for phage infection. Phage adsorption assays showed that the production of EPS does not confer a significant phage resistance phenotype.

The symmetric part of the 31P NMR chemical shielding tensor was determined in a single crystal of 2‐aminoethylphosphonic acid (AEP). The principal values, relative to 85% H3PO4, are σ11=87.1, σ22=15.6, and σ33=−47.5 ppm. Four orientations of the chemical shielding tensor on the molecule are possible, but only one of them shows correlation with the bond directions. The two most shielded components of the tensor lie almost in the plane containing the two P=O bonds, with σ22 approximately bisecting the O–P–O angle and σ33 roughly along the O–O connecting vector, leaving the phosphorus relatively deshielded in the perpendicular σ11 direction. The results can be correlated with the electron density distribution around the phosphorus atom, as determined by the bonding pattern of the phosphonate. A comparison is made with 31P NMR chemical shielding tensors in analogous phosphorus‐containing compounds.

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3′ degenerate core based on four highly conserved amino acids and a longer 5′ consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus , L. casei , Lactobacillus zeae , and Streptococcus thermophilus . The priming GT gene was detected in the genome of both non-EPS-producing (EPS − ) and EPS-producing (EPS + ) strains of L. rhamnosus . The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS + bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS + strains of L. rhamnosus . Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.