Thesis (doctoral)--Rijksuniversiteit te Utrecht.

Dissertation (PhD)--University of Stellenbosch, 2005. / Author's name on university's graduation list: Gundllapalli Moses. Bibliography. Also available via the Internet.

The chemical synthesis of sulfate and phosphate derivatives of galactose-α-1,4-N-acetylglucosamine-OR (octyl N-acetyllactosamine) and galactose-α-1,3-N- acetylglucosamine-OR [where R= -(CH2)7CH3] are reported here using N-acetylglucosamine and galactose as starting materials. Sialylation of octyl N- acetyllactosamine derivatives was achieved using trans-sialidase. These compounds were evaluated as potential acceptor substrates for five recombinant α-1,3- fucosyltransferases (α-1,3-FucT) and a semi-pure α-1,3/4-FucT (human milk). The kinetic data showed a wide range of acceptor specificities between different recombinant enzymes. Octyl N-acetyllactosamine 6-O-sulfate proved to be an excellent substrate for α-1,3-FucT VI, with a KM of 0.85 ?M. This substrate has a lower KM than any reported substrate for any α-1,3-FucT. An unusual result was observed for octyl N- acetyllactosamine derivatives containing a sulfate or phosphate group at the site of glycosylation. These compounds were found to be good acceptor substrates for α-1,3-FucT VI and milk α-1,3/4-FucT with KM and Vmax values similar to those of the parent compound, octyl N-acetyllactosamine. Preliminary studies show that the product of such a reaction could contain a sulfate or phosphate diester linkage between fucose and octyl LacNAc. If the anionic substituent at the site of glycosylation is being fucosylated, current models proposed for a mechanism involving an enzyme active site base mechanism cannot explain this result. An alternative mechanism has been suggested involving Mn2+ coordination to the hydroxyl group of the acceptor substrate being glycosylated. This mechanism can also be used to explain the unusual kinetic results obtained for substrates containing a sulfate or phosphate group at the site of glycosylation.

Medicinal mushrooms are therapeutic agents in traditional folk medicines. Previous studies have shown that a number of biologically active compounds in medicinal mushrooms contributed therapeutic functions against many diseases. These compounds include mainly large molecular weight (MW) compounds such as polysaccharides, dietary fibre and lipids. Mushroom polysaccharides have attracted great attention in food and pharmacology fields because of their biological activities. Polysaccharides vary in molecular weight, degree of branching and conformational structure. It has been reported that fine structure, molecular weight, and conformation of polysaccharide influence biological activities. The incidence and prevalence of inflammatory bowel disease (IBD) have been increasing worldwide, which is characterized by chronic inflammation of the gastrointestinal tract but without satisfactory treatment. Although there are many studies for the immuno-pharmacological activity of mushroom polysaccharides, their intestinal anti-inflammatory property has not been investigated sufficiently. Therefore, it is very important to elucidate whether there is the relationship among the MW, structure and anti-inflammatory activity of polysaccharide in IBD. Firstly, an exopolysaccharide from a mycelial culture of S. commune was obtained by isolation and purification using DEAE-52 cellulose and Sephadex G-150 column chromatography. The structure, conformation and chemical properties were investigated, including elemental compositions, MW, monosaccharide compositions, fourier transform infrared spectrum, thermogram analysis, nuclear magnetic resonance (NMR) spectrum, circular dichroism (CD) study, methylation analysis, and scanning electron microscope (SEM). The findings indicate that the exopolysaccharide is a homogeneous protein-bound heteropolysaccharide carrying molecular weight of 2900 kDa with a β-type glycosidic linkage. It belongs to a kind of β-(13)-D-glucans consisting of a backbone of β-(13)-linked glucose residues branched with (14) and (16)-β-D-glucopyranosyl residues on main-chain residues. The elemental analysis of this exopolysaccharide discover the element compositions as: C, 25.84%; H, 5.45%; and N, 0.65%. The total carbohydrate, protein and uronic acid contents of exopolysaccharide is 89.0%, 2.20% and 7.52%, respectively. In addition, lipopolysaccharide (LPS) was not detected in the exopolysaccharide. Glucose is the main monosaccharide structural unit in this exopolysaccharide, the content is 57.5%. The degradation temperature of exopolysaccharide is 278.9°C from the thermogram analysis curve. This exopolysaccharide looks like thin film with smooth and glittering surface in SEM photography. It is clear from these images that the exopolysaccharide is linear in structure and branched and coiled in aqueous solution. With these extraction, the preliminary anti-inflammatory activity of S. commune exopolysaccharide was conducted by inhibiting the production of nitric oxide (NO), activity of inducible nitric oxide synthase (iNOS) and activity of 5-lipoxygenase (5-LOX) from RAW 264.7 macrophages. The results showed that exopolysaccharide significantly inhibit LPS-induced iNOS expression levels in a dose-dependent manner(p < 0.05). It inhibits the production of 5-LOX in cells, but not in dose-dependence. Further, in dextran sulfate sodium (DSS)-induced colitis model, the results showed that exopolysaccharide attenuated body weight loss, diarrhea, fecal blood, and the shortening of colon and improved histological changes. Furthermore, exopolysaccharide treatment would reduce NO production and some cytokines' secretion such as IL-4 and IL-17A. These results indicate that exopolysaccharide might be exploited as an effective anti-inflammatory agent for application in IBD. Secondly, ultrasound technology was applied to modify the physicochemical properties (MW and viscosity) of this fungal exopolysaccharide, and fractions of different MWs were obtained through ultrasonic degradation method. Effect of the MW degradation, viscosity and anti-inflammatory property of exopolysaccharide under ultrasonic treatment were optimized with response surface methodology. The best ultrasonic treatment parameters were obtained with a three-variable-three-level Box-Behnken design. The optimized conditions for efficient anti-inflammatory activity include: Initial concentration - 0.4%; ultrasonic power - 600 W; and duration of ultrasonic treatment - 9 min. Under these conditions, the NO inhibition rate is 95 ± 0.03% which agreed closely with the predicted value (96%). Average MW of exopolysaccharide decreased after ultrasonic treatments, but no significant change in the preliminary structure by infrared spectroscopy analysis. The viscosity of degraded exopolysaccharide dropped compared with native exopolysaccharide. The results suggest that ultrasound technology is an effective approach to reduce the MW of exopolysaccharide. Our results also showed that exopolysaccharide from S. commune was degraded into three fractions (low, medium, and high MW) by ultrasonic treatment. The changes of MW, atomic force microscope morphology, X-ray diffraction, particle size distribution and viscosity analysis indicate the triple helical structure of exopolysaccharide was dissociated into single helical structure and random coiled structure by breaking of inter- and intramolecular hydrogen bonds. The medium and high MW exopolysaccharide had the mixture of triple helix and single helix conformation. Moreover, the low MW exopolysaccharide exhibit random coiled conformation. As for their anti-inflammatory effect in DSS-induced colitis mice model, the results showed that medium and high MW exopolysaccharide significantly recovered DSS-induced colitis in body weight loss, shortening of colon lengths, colon weight loss, diarrhea and rectal bleeding, histological score, myeloperoxidase (MPO) activity, NO and cytokines (IFN-γ, IL-10 and IL-17) production in inflamed tissues. Moreover, exopolysaccharide with medium and high MW reduced DSS-induced infiltration of macrophages. These results showed that medium and high MW exopolysaccharide had intestinal anti-inflammatory activity. The degraded exopolysaccharide with medium and high MW had a triple and single-helical structure. These results suggested that the intestinal anti-inflammatory activity of exopolysaccharide from S. commune is related to both helical structure and MW.

>Magister Scientiae - MSc / Fucoidan is a marine-derived sulphated polysaccharide with bioactive properties ideal for the food, chemical and pharmaceutical industries. The polysaccharide consists largely of L-fucose, has a highly heterogeneous structure and is of diverse origin. Fucoidan was extracted from Ecklonia maxima, Laminaria pallida and Splachnidium rugosum and the effect of different extraction methods on fucoidan heterogeneity was assessed. Extraction methods employed hot water, hydrochloric acid or calcium chloride salt. Fucoidan yield and purity were determined by various colorimetric assays. Highest fucoidan yield was obtained with the hot water extraction method as seen by highest L-fucose content. Splachnidium rugosum extracts contained ~5 times more L-fucose than Ecklonia maxima and Laminaria pallida extracts. The salt extraction method yielded extracts free of contaminants, however L-fucose content in all extracts was >20 times lower. Acid extraction yielded highest levels of uronic acid contamination and liberated sulphate from the fucoidan polysaccharide. The fucose-to-sulphate ratio for Ecklonia maxima was approximately 1:5, whilst the ratios for Splachnidium rugosum and Laminaria pallida were approximately 1:1 and 1:2, respectively. The acid and salt extraction methods removed all traces of protein contaminants, while the hot water method retained very low levels of protein. The extraction method used to isolate fucoidan was a determining factor in yield and purity. Chemical compositional analyses of hot water extracts were assessed by gas chromatography mass spectroscopy. Splachnidium rugosum and Laminaria pallida extracts consisted largely of L-fucose, while Ecklonia maxima fucoidan was characterized with high glucose abundance. Crude hot water and acid extracts from Splachnidium rugosum tissue were fractionated and purified by (anionic) ion exchange chromatography as bioactivity has been correlated to lower molecular weight forms. In water extracts, ion exchange chromatography resulted in close to 90% decrease in L-fucose, sulphate and uronic acid, while protein content increased by 57%. Similar results were reported for acid extracts; however protein content did not change significantly. These results show that method of extraction may affect the composition of fucoidan post-purification. Hot water extraction is recommended due to higher fucoidan yield, as reflected by L-fucose content, and higher sulphate-to-fucose ratio. High protein content after ion exchange chromatography was however of concern. Since mucilage in Splachnidium rugosum thallus was free of protein, fucoidan was precipitated from mucilage with ethanol. Fucoidan yield of mucilage was >15-fold higher than content in purified hot water extracts with a sulphate-to-fucose ratio of ~1:1. The average molecular weight of native fucoidan in mucilage was estimated at 2367 kDa. The polysaccharide was hydrolysed by gamma-irradiation levels of 10-50 kGy to fractions ranging between 60 and 15.5 kDa. Hot water crude fucoidan extracts from Ecklonia maxima, Laminaria pallida, and Splachnidium rugosum were assessed for anti-oxidant activity by measuring the ability to scavenge free radicals and the capacity to reduce copper ions with 2,2-Diphenyl-1-picrylhydrazyl and Cupric Reducing Anti-oxidant Capacity assays, respectively. Ecklonia maxima crude fucoidan displayed highest anti-oxidant activity and capacity, having the potential to scavenge reactive oxygen species as well as the capacity to reduce copper to less toxic forms in mammalian systems. Splachnidium rugosum showed weakest anti-oxidant activity and lowest reducing capacity. The anti-cancer activity of crude and purified hot water Splachnidium rugosum extracts, as well as non-irradiated (native) and gamma-irradiated fucoidan, and commercially procured fucoidan were assessed for anti-cancer activity against MCF-7 breast cancer cells. Splachnidium rugosum crude and purified fucoidan displayed a half maximal inhibitory concentration of 0.7 mg/mL and 0.029 mg/mL, respectively. Low cytotoxicity of crude and purified Splachnidium rugosum fucoidan against non-cancerous breast epithelial cell line MCF-12A was observed, as seen by half maximal inhibitory concentration values of 2 mg/mL and 0.663 mg/mL, respectively. The cancer specific selectivity of purified Splachnidium rugosum fucoidan was therefore much higher as reflected by 10-fold higher selectivity index than that of crude fucoidan. Native and low molecular weight gamma-irradiated fucoidan also showed bioactive properties including anti-cancer activity as seen by the reduction of cell proliferation in vitro, whereas crude fucoidan showed the ability to scavenge free radicals, and the capacity to reduce copper ions. / National Research Foundation (NRF)

No description available.

The work described in this thesis forms part of an international programme concerned with the structure elucidation of the capsular antigens of some Enterobacteriaceae. Many of the Klebsiella and some of the Escherichia coli are pathogenic to man and, hence, they are of interest. The virulence of bacteria is a multifactorial phenomenon, in which characteristic traits of bacteria and their hosts play comparable and complementary roles. It is accepted that pathogens are more virulent when encapsulated, because, nearly all disease causing bacteria have a capsule when freshly isolated from the host. This increase in pathogenicity is related, in part, to the capsular polysaccharides' ability to avoid or attenuate the host defence mechanisms. In the majority of cases the protective aspects of the capsule are overcome in the latter stages of infection when the formation of specific antibodies by the host has occurred. However there are situations in which an immune state of the infected host is virtually never reached, and susceptiblity to the infecting bacteria is maintained even in the advanced stage of an infection. Explanation of this phenomenon becomes possible by analysing the structure of the polysaccharides. The inability of the host to raise an immune response to the capsule may be because the structure of the polysaccharide is similar or identical to the host's carbohydrates. The serological and pathogenic relatedness of encapsulated E. coli and Klebsiella, to the encapsulated strains of other genera, is based on structural identity or similarity of the respective capsules. Capsular polysaccharides are analysed by both chemical and instrumental methods, and, at present, nuclear magnetic resonance spectroscopy is the most important analytical technique

The highly sulphated, methylated polysaccharide isolated from Pachymenia Carnosa, a red seaweed, was shown to contain D- galactose, 2-o (underscore) methyl-D- galactose, 6-o (underscore) -methyl- D- galactose and 4-o (underscore)-methylgalactose. The polysaccharide was desulphated with methanolic hydrogen chloride. Methylation of the desulphated polysaccharide revealed that it was composed entirely of (1→73) and (1→4) links in approximately equal amounts. Treatment of the polysaccharide with alkali showed that the majority of the ester sulphate groups were alkali-stable. Partial hydrolysis and acetolysis studies indicated that the polysaccharide was extremely complex, and contained alternate (1→3) and β (1→4) glycosidic linkages. There is evidence for the presence of D-galactose-6-sulphate.

This thesis presents a detailed chemical and spectroscopic determination of the capsular, polysaccharide K-antigen isolated from the Klebsiella bacterium, serotype K43 (culture #2482). The repeating unit of the capsular polysaccharide was found to be of the "3 + 2" repeating unit type. A uronic acid was found as part of a disaccharide side chain and the main chain of the polysaccharide was found to be composed of a neutral trisaccharide of mannose and galactose. The work forms part of an ongoing research interest in bacterial polysaccharides of this laboratory and now completes the structural elucidation of all the Klebsiella K-antigens, bar three antigens which were originally assigned to other laboratories. These data together with the respective serological characteristics of each serotype are available to the molecular biologist, and may result in the production of: vaccine(s) against Klebsiella infections, diagnostic products and novel carrier molecules enabling targeted drug delivery.

Aeodes ulvoidea, a red seaweed of the Grateloupiaceae, yielded a highly sulphated polysaccharide which was shown to contain D- and L-galactose, 4-0-methy-L-galactose, 2-0-methyl - D- and L-galactose and 6-0-methyl-D-galactose, together with chromatographic traces of xylose and mannose. The sulphate was not labile to alkali, but it was largely removed with methanolic hydrogen chloride. Periodate oxidation of the polysaccharide, methylation of the de sulphated polysaccharide, and investigation of fifteen oligosaccharides from partial hydrolysis and acetolysis studies of the polysaccharide, indicate that (a) the polysaccharide is composed of a backbone of D-galactose residues which are 1,3- and 1,4-linked (b) at least some regions of alternating structure do occur (c) the 2-0-methylgalactose is linked through the 4-position (d) the 4-0-methyl-L-galactose is present as single unit side chains glycosidically linked to the galactose backbone at position 6, and (e) most of the 6-0-methyl-D-galactose is linked to the 4-position of 2-0-methyl-D-galactose.