Pharmazeutische Biologie und Phytochemie. Glycoconjugates from plants as antiadhesive Compounds against Helicobacter pylori

Transcription

1 WESTFÄLISCHE WILHELMS-UNIVERSITÄT MÜNSTER Pharmazeutische Biologie und Phytochemie Glycoconjugates from plants as antiadhesive Compounds against Helicobacter pylori Ribes nigrum L. Abelmoschus esculentus (L.) MOENCH Inaugural-Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften im Fachbereich Chemie und Pharmazie der Mathematisch-Naturwissenschaftlichen Fakultät der Westfälischen Wilhelms-Universität Münster vorgelegt von Jutta Messing aus Bocholt

2 TABLE OF CONTENTS TABLE OF CONTENTS 1 I. LIST OF ABBREVIATIONS 7 II. SUMMARY Introduction Helicobacter pylori History and discovery of the bacterium Microbiology Transmission Prevalence and epidemiology Colonization Outer membrane proteins (OMPs) The blood group antigen binding adhesin (BabA) The sialic acid binding adhesin (SabA) Pathogenesis Diagnosis, treatment and recurrence Alternative treatments Ribes nigrum L Abelmoschus esculentus (L.) MOENCH Aim of this work Results Isolation of the F2 fraction from Ribis nigri semen Isolation of the raw Polysaccharides Isolation of the F2 fraction from Ribis nigri semen RPS by ion-exchange chromatography (IEC) Analytical investigations on the carbohydrate part of F Carbohydrate content of fraction F Uronic acid content of fraction F Arabinogalactan protein (AGP) content of fraction F Molecular weight determination by GPC on Superose Molecular weight determination of F2 by HP-SEC SDS-PAGE Determination of monosaccharide composition of F2 by HPAEC-PAD 49 1

3 Determination of D-/L-configuration of monosaccharides by capillary zone electrophoresis (CZE) Linkage analysis of partially methylated alditol acetates (PMAA) Analytical investigations on the protein part of F Protein content offraction F Determination of the amino acid composition offraction F2 by HPAEC-PAD Distribution of carbohydrates and proteins in F Antiadhesive activity of F2 against H. pylori Preparations before activity testing Identification of H. pylori Correlation of optical density (OD) and colony forming units (CFU) Determination of direct cytotoxicity towards H. pylori Influence of F2 towards the adhesion of H. pylori to human gastric mucosa tissue sections Quantitative in vitro flow cytometric adhesion assay Influence of F2 towards AGS cell viability interaction and binding properties offraction F2 towards H. pylori Dot blot overlay assay Hemagglutination assay Investigation on binding properties and interaction of F2 and okra extract with BabA and SabA by Radioimmunoassay (RIA) Radiolabeling of F2 and okra extract with 12S I Binding of F2 and okra extract to different H. pylori strains Influence of F2 and okra extract on H. pylori binding to Lewis b Influence of F2 and okra extract on H. pylori binding to sialyl-lewis" Further characterization of F Hydrolytic degradation experiments Enzymatic degradation experiments Treatment of F2 with endo-arabinanase and a-l-arabinofuranosidase Treatment of F2 with endo-ß- 1,6-galactanase Treatment of F2 with ß-glucuronidase Treatment of F2 with ß-xylanase Activity testing of F2 subfractions and subfractions derived from ß-glucuronidase treatment 12o 2.9. Elucidation of the Potential antiadhesive activity of glucuronic acid 125

4 Adhesion assay with carbohydrate coupled Polymerie probes Adhesion assay with magnetic nanoparticles Discussion Structural characterization of F Antiadhesive activity of F2 and okra extract The role of glucuronic acid in antiadhesive Compounds Materials and Methods Instruments and consumables Laboratory equipment and instruments Consumable goods Chemicals and reagents General chemicals and reagents Carbohydrates and amino acids Chemicals and reagents for microbiology, cell culture and molecular biology Buffers, solutions and media Periodate treatment of BSA and preparation of blocking buffer IV Plant material and test Compounds Plant material Test Compounds Cell lines Human stomach tissue sections Bacterial strains Isolation of Polysaccharides from Ribis nigri semen Isolation of the raw Polysaccharides from Ribis nigri semen Isolation ofthe F2 fraction from Ribis nigri semen RPS Preparation of okra extract Analytical methods Colorimetric methods Determination of the total carbohydrate content in F2 according to Monsigny et al. (1988) Determination of the total uronic acid content in F2 according to Blumenkrantz and Asboe-Hansen (1973) with modifications according to Kram (1984) Determination ofthe total protein content in F Semi-quantitative detection of arabinogalactan proteins according to van Holst et al. (1985) 171

5 4 TABLE OF CONTENTS Hydrolysis, reduction, derivatization and partially degradation Hydrolysis of Polysaccharides with trifluoroacetic acid (TFA) Hydrolysis of Polypeptides with HCl (6 Mol/L) Hydrolysis of Polypeptides with NaOH (4.2 Mol/L) Derivatization for determination of D-/L-configuration according to Noe and Freissmuth (1995) Reduction of glycoproteins according to Taylor et al. (1972) Methylation analysis of glycoproteins according to Hakomori (1964) with modification according to Harris (1984) Hydrolytic degradation, with additional ethanol precipitation Enzymatic degradation Semi-purification of Onozuka RIO cellulase according to Okemoto et al. (2003) Chromatographie methods lon-exchange chromatography (IEC) Gel-permeation chromatography (GPC) High-performance size-exclusion chromatography (HP-SEC) High-performance anion-exchange chromatography with pulsed-amperometric detection (HPAEC-PAD) Gas chromatography (GC) Electrophoretic methods Capillary zone electrophoresis (CZE) for determination of D-/L-configuration of carbohydrates Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (1970) , Detection of AGPs in SDS-PAGE by Yariv reagent staining Detection of proteins in SDS-PAGE by silver staining i Dialysis Methods for cell culture and microbiology General working procedures Sterilization Working procedures for cell culture Defrosting of AGS cells Cultivation of AGS cells Passaging of AGS cells Cryopreservation of AGS cells 191

6 Calculation of cell density Working procedures for microbiology Cultivation of H. pylori Cryopreservation of H. pylori Correlation of optical density (OD) and colony forming units (CFU) Identification of H. pylori Isolation of bacterial DNA DNA quantification Real-time Polymerase chain reaction (PCR) Primers for real-time PCR FITC labeling of H. pylori Determination of cell viability and cytotoxicity, MTT-assay (Mosmann 1983) Agar diffusion test Adhesion assay of H. pylori with human gastric tissue sections Quantitative In vitro flow cytometric adhesion assay of H. pylori with AGS cells Adhesion assay of H. pylori with AGS cells determined by fluorescence intensity Dot blot overlay assay Hemagglutination assay H. pylori adhesin interaction studies by radioimmunoassay (RIA) Radiolabeling of glycoproteins with the chloramine T method according to Hunter and Greenwood (1962) Radioimmunoassay (RIA) with H. pylori Determination of binding properties of F2 and okra extract towards different H. pylori strains Determination of inhibitory effects of F2 and okra extract towards Lewis b binding of H. pylori Determination of inhibitory effects of F2 and okra extract towards sialyl-lewis* binding of H. pylori Adhesion assay of H. pylori to carbohydrate coupled Polymerie probes Adhesion assay of H. pylori to magnetic nanopartides Statistics Software 211 III. REFERENCES 212

7 PUBUCATIONS 238 CURRICULUM VITAE 239