Evaluation of Macroalgae Sulfated Polysaccharides on the Leishmania (L.) amazonensis Promastigote

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Polysaccharides as safer release systems for agrochemicals

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Immediate and long-term effects of polysaccharides-based formulations on human skin

A new trend in cosmetic formulations is the use of biotechnological raw materials as the polysaccharides from Klebsiella pneumoniae, which are supposed to enhance cell renewal, improve skin hydration and micro-relief. Botanical extracts of Myrtus communis leaves contain different sugars, which may provide the same benefits. Thus, the objective of this study was to evaluate through objective and subjective analysis the immediate and long-term effects of cosmetic formulations containing polysaccharides biotechnologically-originated and / or the ones contained in Myrtus communis extracts. Three polysaccharide-based and placebo formulations were applied on the forearm skin of 40 volunteers. Skin hydration, transepidermal water loss (TEWL), viscoelasticity and skin micro-relief measurements were made before and 2 hours after a single application and after 15 and 30 day-periods of daily applications. Answers to a questionnaire about perceptions of formulation cosmetic features constituted the subjective analysis. All polysaccharide-based formulations enhanced skin hydration. Formulations with isolated or combined active substances improved skin barrier function as compared to placebo, in the short and long term studies. Formulations containing Myrtus communis extracts had the highest acceptance. Results suggest that daily use of formulations containing these substances is important for protection of the skin barrier function.

Synthesis, structure and conformation of oligo- and polysaccharides

Carbohydrates are a complex group of biomolecules with a high structural diversity. Their almost omnipresent occurrence has generated a broad field of research in both biology and chemistry. This thesis focuses on three different aspects of carbohydrate chemistry, synthesis, structure elucidation and the conformational analysis of carbohydrates. The first paper describes the synthesis of a penta- and a tetrasaccharide related to the highly branched capsular polysaccharide from Streptococcus pneumoniae type 37. In the second paper, the structure of the O-antigenic repeating unit from the lipopolysaccharide of E. coli 396/C1 was determined along with indications of the structure of the biological repeating unit. In addition, its structural and immunological relationship with E. coli O126 is discussed. In the third paper, partially protected galactopyranosides were examined to clarify the origin of an intriguing 4JHO,H coupling, where a W-mediated coupling pathway was found to operate. In the fourth paper, the conformation of methyl a-cellobioside is studied with a combination of molecular dynamics simulations and NMR spectroscopy. In addition to the expected syn-conformation, detection and quantification of anti-ø and anti-ψ conformers was also possible.

Structural and Interaction Studies of Bacterial Polysaccharides by NMR Spectroscopy

An introduction to bacterial polysaccharides and the methods for structural determination are described in the first two parts of the thesis. In a structural elucidation of bacterial polysaccharides NMR experiments are important as is component analysis. A short description of immunochemical methods such as enzyme immunoassays is included. Two NMR techniques used for interaction studies, trNOE and STD NMR, are also discussed. The third part of the thesis discusses and summarizes the results from the included papers. The structures of the exopolysaccharides produced by two lactic acid bacteria are determined by one- and two dimensional NMR experiments. One is a heteropolysaccharide produced by Streptococcus thermophilus and the other a homopolysaccharide produced by Propionibacterium freudenreichii. The structure of an acidic polysaccharide from a marine bacterium with two serine residues in the repeating unit is also investigated. The structural and immunological relationship between two O-antigenic polysaccharides from Escherichia coli strain 180/C3 and O5 is discussed and investigated. Finally, interaction studies of an octasaccharide derived from the Salmonella enteritidis O-antigen and a bacteriophage are described which were performed with NMR experiments.

Characterization of polysaccharides from Hypnea spinella (Gigartinales) and Halopithys incurva (Ceramiales) and their effect on RAW 264.7 macrophage activity

[EN] Red algae have been reported to be an important source of polysaccharides with potential immunomodulatory properties. The objective of this study was to characterize the polysaccharides from Halopithys incurva and Hypnea spinella and to evaluate their effect on the synthesis of cytokines by murine cell line RAW 264.7 macrophages. Polysaccharides were obtained by N-cetylpyridiniumbromide precipitation and characterized by Fourier transform-infrared spectroscopy. Their effect on the activity of RAW 264.7 macrophages was examined by quantification of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and nitric oxide (NO) production using enzyme-linked immunosorbent assays. The activation of the cytokine IL-6 and NO increased linearly as the concentration of polysaccharides from H. incurva and Hy. spinella increased. In general, the activation of IL-6 and NO was tenfold greater when macrophages were exposed to polysaccharides from H. incurva than when exposed to polysaccharides from Hy. spinella. In contrast, TNF-α concentration did not increase when macrophages were exposed to increasing polysaccharide levels. These results indicate that polysaccharides are strong cytokine IL-6 inducers.

The immunogenicity of chemically derived zwitterionic polysaccharides

Bacterial capsular polysaccharides (PS) which naturally contain zwitterionic charge motifs (ZPS) possess specific immunostimulatory activity, leading to direct activation of antigen-presenting cells (APCs) through Toll-like receptor 2 (TLR2) and of T cells in co-culture systems. When administered intraperitoneally, ZPS and bacteria expressing them are involved in the induction or regulation of T-cell dependent inflammatory processes such as intra-abdominal abscess formation. Moreover it has been published that ZPSs are processed to low molecular weight carbohydrates and presented to T cells through a pathway similar to that used for protein antigens. These findings were in contrast with the paradigm according to which polysaccharides are T-independent antigens unable to be presented in association with MHC class II molecules and unable to induce a protective immune response. For this reason in glycoconjugate vaccines polysaccharides often need to be conjugated to a carrier protein to induce protection. The aim of our work was to generate vaccine candidates with antigen and adjuvant properties in one molecule by the chemical introduction of a positive charge into naturally anionic PS from group B streptococcus (GBS). The resulting zwitterionic PS (ZPS) has the ability to activate human and mouse APCs, and in mixed co-cultures of monocytes and T cells, ZPS induce MHC II-dependent T-cell proliferation and up-regulation of activation markers. TLR2 transfectants show reporter gene transcription upon incubation with ZPS and these stimulatory qualities can be blocked by anti-TLR2 mAbs or by the destruction of the zwitterionic motif. However, in vivo, ZPS used alone as vaccine antigen failed to induce protection against GBS challenge, a result which does not confirm the above mentioned postulate that ZPS are T-cell dependent Ags by virtue of their charge motif. Thus to make ZPS visible to the immune system we have conjugated ZPS with a carrier protein. ZPS-glycoconjugates induce higher T cell and Ab responses to carrier and PS, respectively, compared to control PS-glycoconjugates made with the native polysaccharide form. Moreover, protection of mothers or neonate offspring from lethal GBS challenge is better when mothers are immunized with ZPS-conjugates compared to immunization with PS-conjugates. In TLR2 knockout mice, ZPS-conjugates lose both their increased immunogenicity and protective effect after vaccination. When ZPS are co-administered as adjuvants with unconjugated tetanus toxoid (TT), they have the ability to increase the TT-specific antibody titer. In conclusion, glycoconjugates containing ZPS are potent vaccines. They target Ag to TLR2-expressing APCs and activate these APCs, leading to better T cell priming and ultimately to higher protective Ab titers. Thus, rational chemical design can generate potent novel PS-adjuvants with wide application, including glycoconjugates and co-administration with unrelated protein Ags.

Synthesis of oligosaccharides corresponding to the polysaccharides of Lactobacillus and Thermophlilus strains

Synthesis of a branched trisaccharide and a tetrasaccharide repeating units corresponding to the polysaccharides of Lactobacillus spp. G-77 and Thermus thermophilus Samu-SA1 as their methyl glycosides has been achieved in excellent yield. Most of the glycosyl linkages are 1,2-cis in these oligosaccharide fragments

Further characterization of the epa gene cluster and Epa polysaccharides of Enterococcus faecalis.

We previously identified a gene cluster, epa (for enterocococcal polysaccharide antigen), involved in polysaccharide biosynthesis of Enterococcus faecalis and showed that disruption of epaB and epaE resulted in attenuation in translocation, biofilm formation, resistance to polymorphonuclear leukocyte (PMN) killing, and virulence in a mouse peritonitis model. Using five additional mutant disruptions in the 26-kb region between orfde2 and OG1RF_0163, we defined the epa locus as the area from epaA to epaR. Disruption of epaA, epaM, and epaN, like prior disruption of epaB and epaE, resulted in alteration in Epa polysaccharide content, more round cells versus oval cells with OG1RF, decreased biofilm formation, attenuation in a mouse peritonitis model, and resistance to lysis by the phage NPV-1 (known to lyse OG1RF), while mutants disrupted in orfde2 and OG1RF_163 (the epa locus flanking genes) behaved like OG1RF in those assays. Analysis of the purified Epa polysaccharide from OG1RF revealed the presence of rhamnose, glucose, galactose, GalNAc, and GlcNAc in this polysaccharide, while carbohydrate preparation from the epaB mutant did not contain rhamnose, suggesting that one or more of the glycosyl transferases encoded by the epaBCD operon are necessary to transfer rhamnose to the polysaccharide. In conclusion, the epa genes, uniformly present in E. faecalis strains and involved in biosynthesis of polysaccharide in OG1RF, are also important for OG1RF shape determination, biofilm formation, and NPV-1 replication/lysis, as well as for E. faecalis virulence in a mouse peritonitis model.

Ozonolysis for selectively depolymerizing polysaccharides containing β-d-aldosidic linkages

The depolymerization of polysaccharides, particularly those containing acid-sensitive components, into intact constituent repeating units can be very difficult. We describe a method using ozonolysis for depolymerizing polysaccharides containing β-d-aldosidic linkages into short-chain polysaccharides and oligosaccharides. This method is carried out on polysaccharides that have been fully acetylated whereby β-d-aldosidic linkages are selectively oxidized by ozone to form esters, from which the polysaccharides are subsequently cleaved with a nucleophile. Ozone oxidation of aldosidic linkages proceeds under strong stereoelectronic control, and reaction rates depend on the conformations of glycosidic linkages. Thus, β-d-aldosidic linkages with different conformations can have very different reaction rates even in the absence of substantial chemical differences. These rate differences allowed for very high selectivity in cleaving β-d-linkages of polysaccharides. Several polysaccharides from group B Streptococcus and other bacterial species were selectively depolymerized with this method. The repeating units of the group B Streptococcus polysaccharides all contain an acid-sensitive sialic acid residue in a terminal position on a side chain and several β-d-residues including galactose, glucose, and N-acetylglucosamine; however, with each polysaccharide, one type of linkage was more reactive than others. Selective cleavage of the most sensitive linkage occurs randomly throughout the polymer chain, yielding fragments of controllable and narrowly distributed sizes and the same repeating-unit structure. The average size of the molecules decreases exponentially, and desired sizes can be obtained by stopping the reaction at appropriate time points. With this method the labile sialic acid residue was not affected.

A Cross-Polarization, Magic-Angle-Spinning, 13C-Nuclear-Magnetic-Resonance Study of Polysaccharides in Sugar Beet Cell Walls1

Solid-state nuclear magnetic resonance relaxation experiments were used to study the rigidity and spatial proximity of polymers in sugar beet (Beta vulgaris) cell walls. Proton T1ρ decay and cross-polarization patterns were consistent with the presence of rigid, crystalline cellulose microfibrils with a diameter of approximately 3 nm, mobile pectic galacturonans, and highly mobile arabinans. A direct-polarization, magic-angle-spinning spectrum recorded under conditions adapted to mobile polymers showed only the arabinans, which had a conformation similar to that of beet arabinans in solution. These cell walls contained very small amounts of hemicellulosic polymers such as xyloglucan, xylan, and mannan, and no arabinan or galacturonan fraction closely associated with cellulose microfibrils, as would be expected of hemicelluloses. Cellulose microfibrils in the beet cell walls were stable in the absence of any polysaccharide coating.

Borate-Rhamnogalacturonan II Bonding Reinforced by Ca2+ Retains Pectic Polysaccharides in Higher-Plant Cell Walls1

The extent of in vitro formation of the borate-dimeric-rhamnogalacturonan II (RG-II) complex was stimulated by Ca2+. The complex formed in the presence of Ca2+ was more stable than that without Ca2+. A naturally occurring boron (B)-RG-II complex isolated from radish (Raphanus sativus L. cv Aokubi-daikon) root contained equimolar amounts of Ca2+ and B. Removal of the Ca2+ by trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid induced cleavage of the complex into monomeric RG-II. These data suggest that Ca2+ is a normal component of the B-RG-II complex. Washing the crude cell walls of radish roots with a 1.5% (w/v) sodium dodecyl sulfate solution, pH 6.5, released 98% of the tissue Ca2+ but only 13% of the B and 22% of the pectic polysaccharides. The remaining Ca2+ was associated with RG-II. Extraction of the sodium dodecyl sulfate-washed cell walls with 50 mm trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid, pH 6.5, removed the remaining Ca2+, 78% of B, and 49% of pectic polysaccharides. These results suggest that not only Ca2+ but also borate and Ca2+ cross-linking in the RG-II region retain so-called chelator-soluble pectic polysaccharides in cell walls.

Novel, Starch-Like Polysaccharides Are Synthesized by an Unbound Form of Granule-Bound Starch Synthase in Glycogen-Accumulating Mutants of Chlamydomonas reinhardtii1

In vascular plants, mutations leading to a defect in debranching enzyme lead to the simultaneous synthesis of glycogen-like material and normal starch. In Chlamydomonas reinhardtii comparable defects lead to the replacement of starch by phytoglycogen. Therefore, debranching was proposed to define a mandatory step for starch biosynthesis. We now report the characterization of small amounts of an insoluble, amylose-like material found in the mutant algae. This novel, starch-like material was shown to be entirely dependent on the presence of granule-bound starch synthase (GBSSI), the enzyme responsible for amylose synthesis in plants. However, enzyme activity assays, solubilization of proteins from the granule, and western blots all failed to detect GBSSI within the insoluble polysaccharide matrix. The glycogen-like polysaccharides produced in the absence of GBSSI were proved to be qualitatively and quantitatively identical to those produced in its presence. Therefore, we propose that GBSSI requires the presence of crystalline amylopectin for granule binding and that the synthesis of amylose-like material can proceed at low levels without the binding of GBSSI to the polysaccharide matrix. Our results confirm that amylopectin synthesis is completely blocked in debranching-enzyme-defective mutants of C. reinhardtii.

Natural Pectin Polysaccharides as Edible Coatings

The most fashionable trends in food packaging research are targeted towards improvements in food quality and safety by increasing the use of environmentally-friendly materials, ideally those able to be obtained from bio-based resources and presenting biodegradable characteristics. Edible films represent a key area of development in new multifunctional materials by their character and properties to effectively protect food with no waste production. The use of edible films should be considered as a clean and elegant solution to problems related with waste disposal in packaging materials. In particular, pectin has been reported as one of the main raw materials to obtain edible films by its natural abundance, low cost and renewable character. The latest innovations in food packaging by the use of pectin-based edible films are reviewed in this paper, with special focus on the use of pectin as base material for edible coatings. The structure, properties related to the intended use in food packaging and main applications of pectins are herein reported.