Introduction 1.1 Helicobacter pylori and gastroduodenal diseases Helicobacter pylori is a gram-negative, spiral-shaped microaerophilic bacterium that colonizes the human gastric mucosa. Since the successful isolation of H. pylori by Marshall and Warren in 1983, doors have been opened for scientists to study the association of H. pylori with various gastroduodenal diseases. Persistent colonization of H. pylori in human gastrointestinal tracts has been closely linked to gastric diseases ranging from gastritis, non-ulcer dyspepsia and peptic ulcer to the increased risks of gastric cancer (Buck et al., 1986; Dunn et al., 1997). Being a major human gastric pathogen, H. pylori infects more than half of the world’s population and has been causing gastric diseases worldwide (Blaser et al., 1994; Uemura et al., 2001). For the past two decades, great effort has been focused on the study of H. pylori with respect to its bacteriology, physiology, genetics, pathogenesis and epidemiology of infection (Rothenbacher & Brenner, 2003). Treatment of peptic ulcer disease has been developed (Graham et al., 1992). However, current therapies remain to be improved as the organism is perfectly adapted to the ecological niche in the gastric mucosa and currently available antibiotics are not specifically designed to be active in the stomach (Sherwood et al., 2002; Schreiber et al., 2004). 1.2 Characteristics of H. pylori Two morphological forms were observed in H. pylori: spiral and coccoid (Hua & Ho, 1996). The spiral-shaped H. pylori is the active form capable of colonization and infection (Dunn et al., 1997). On the other hand, the coccoid form is considered as viable but non-culturable (Van et al., 1994) and has been considered as the resting state of the Introduction bacterium (Benaissa et al., 1996). Under unfavorable conditions such as depletion of nutrients, addition of antibiotics or stress stimuli (low pH or high temperature), morphological conversion from spiral to coccoid can be observed in in vivo culture (Catrenich & Makin, 1991). However, the resuscitation from coccoid to spiral has not been reported in in vitro conditions. Therefore, controversies remain among researchers as some regarded coccoids as dead bacterial cells (Kusters et al., 1997; Enroth et al., 1999) while others believed that coccoids are viable but non-culturable (Hua & Ho, 1996; Zheng et al., 1999; Saito et al., 2003). The spiral form of H. pylori expresses a great number of proteins that participate in various bacterial metabolic activities such as cell survival and proliferation, adhesion, colonization and transportation of macromolecules. In contrast, the coccoid expresses significantly less proteins related to the basic metabolism such as cell respiration, maintaining cellular integrity and DNA synthesis (Kusters et al., 1997; Narikawa et al., 1997; Costa et al., 1999). It is widely agreed that the spiral form is mainly responsible for the pathogenesis of H. pylori infection (Dubois, 1995). In contrast, it has been suggested that the dormant coccoid form may be involved in the transmission of H. pylori infection (Hua & Ho, 1996; Zheng et al., 1999; Andersen et al., 2000; Ng et al., 2003). 1.3 Pathogenesis of H. pylori Several proteins have been identified in H. pylori to be associated with its virulence and pathogenesis. The most intensively studied virulence factors are cytotoxin-associated immuno-dorminant protein (CagA), vacuolating toxin A (VacA), adhesins, flagella, urease and heat shock proteins (HSPs). These factors act independently from each other Introduction in the course of H. pylori infection but are indispensable for bacterial pathogenesis (Prinz et al., 2003). Among the various virulence factors, adhesins are important for mediating receptorligand recognition in the initial interaction between H. pylori and host. Many H. pylori proteins have been identified as adhesins specific for interaction with particular ligands of the host (Aspholm et al., 2004). These include blood-group-antigen-binding adhesion (BabA) which is an adhesin of H. pylori interacting with the blood group antigen-Lewis antigen on gastric epithelial cells (Ilver et al., 1998; Hennig et al., 2004); Sialic acidbinding adhesion (SabA) that is responsible for the binding of H. pylori to sialyl-Lewis x antigens in gastric epithelium in humans (Mahadavi et al., 2002); OipA (outer inflammatory protein) and HopZ (homologue of porin) which are associated with the adhesion and colonization of H. pylori in vitro and in vivo, respectively (Yamaoka et al., 2002). The sheathed flagellum is responsible for the motility of H. pylori which is necessary for bacterial survival in the viscous mucus layer (Josenhans et al., 1995) while surface localized urease is an enzyme served to maintain a neutral pH microenvironment for the survival of H. pylori in the acidic stomach (Eaton et al., 1991; Perez-Perez et al., 1992; Clyne et al., 1995). CagA and VacA have been proven to be two major virulence factors of H. pylori, of which CagA protein can be translocated into the epithelial cells to trigger a cascade of signal transduction pathways (Segal et al., 1999; Hirata et al., 2004) while VacA is known for its ability to induce cytoplasmic vacuole formation in various eukaryotic cells (Telford et al., 1994; Cover et al., 2005). HSP is another group of Introduction virulence factor essential for maintaining the normal functions of other H. pylori proteins and assisting its survival in the stomach (Kamiya et al., 1998). 1.4 Interaction between H. pylori and gastric mucus layer The human stomach is protected by the gastric acid of a pH around 2.0 while a viscous layer of mucus acts as the protective barrier for the underlying gastric epithelium (Vinall et al., 2002). The main component of the mucus is a high molecular weight glycoprotein known as mucin, which has a long linear structure consisting of subunits joined by disulfide bonds (Thomsson et al., 2002). The subunit of mucin contains a peptide core that is interspersed among clusters of oligosaccharide side chains (Stanley et al., 1983; Karlsson et al., 1996). The diversity of mucin oligosaccharides has been found to provide multiple receptors for bacterial lectins of invading pathogens (Karlsson et al., 1989 & 1995). Furthermore, experimental evidence has illustrated that the adherence to mucin occurs in a number of pathogens such as Pseudomonas aeruginosa, Candida albicans and Staphylococcus aureus (Hoffman et al., 1993; Bruce et al., 1995; Shuter et al., 1996). In the case of H. pylori, it is believed that attachment to the mucus layer has been established between the bacteria and host before its colonization at the epithelium and specific adhesins could exist in mediating such interaction (Linden et al., 2002). Kalpana (2003) described a protein from H. pylori that has the ability of binding to mucin in in vitro assays which has been identified to be a hypothetical protein of H. pylori coded as HP0049 in the genome of H. pylori ATCC 26695 (Tombs et al., 1997). It is therefore interesting to characterize the functions of this novel protein and explore its possible role in the pathogenesis of H. pylori related gastroduodenal diseases. Introduction 1.5 Objectives of study This study aims to characterize this hypothetical protein HP0049 and examine its functions in H. pylori. The main goals of the project are to clone and express the gene encoding HP0049 followed by characterization of the recombinant protein. In the process, the recombinant protein will be employed to raise specific antibody, which will be used for sub-cellular localization of the protein in H. pylori by transmission electron microscopy (TEM). Biochemical and physiological studies will be carried out to explore the probable role of the hypothetical protein HP0049 in H. pylori pathogenesis. Survey of Literature 2.1 Characteristics of H. pylori 2.1.1 Isolation and culturing of H. pylori Research in H. pylori began in earnest after its isolation in 1983 by Marshall and Warren. Helicobacters belong to a new genus of bacteria, mainly inhabiting along the interface of mucosa and gastric epithelial cells of mammals. H. pylori NCTC 11637 is the type species of the genus, Helicobacter. It is gram-negative, microaerophilic, spiralshaped, flagellated and urease positive bacterium. It is a nutritionally fastidious microorganism which is able to form about mm transparent colonies on enriched agar plates supplemented with 5-10% horse blood after 3-5 days of incubation (Goodwin & Worsley, 1993). H. pylori is also an oxygen sensitive bacterium that strictly grows in the presence of 5-10% carbon dioxide at 35-37˚C under humidified conditions (Goodwin et al., 1986). The organism can proliferate in both non-selective and selective media supplemented with antibiotics (Goodwin & Worsley, 1993; Westblom et al., 1991) whereby the latter are widely used for the isolation of H. pylori from biopsy samples. H. pylori can be cultured on solid agar plates as well as in liquid media. However, the growth of H. pylori in liquid media (generally enriched with yeast extract and serum) is comparatively slower than that on the agar plate. Nevertheless, broth culture has been used favourably in the study of its metabolic activities and and physiological properties (Goodwin et al., 1986; Ho & Vijayakumari, 1993). H. pylori has been shown to grow in brain heart infusion (BHI) broth supplemented with 0.4 % yeast extract and 10% horse serum under microaerophilic conditions in an efficient and continuous culture system (Ho & Vijayakumari, 1993). Survey of Literature 2.1.2 Morphological features of H. pylori Based on electron microscopy studies, two major morphological forms of the bacteria were observed: spiral and coccoid. In an early study by Benaissa et al. (1996), the conversion from spiral to coccoid via U-shaped transition form was clearly observed under transmission electron microscopy. Thereafter, similar observations of morphological conversion were reported by other researchers in later studies (Kusters et al., 1997; Costa et al., 1999). The spiral shaped H. pylori is approximately 0.5 µm in width and 2-3 µm in length, equipped with 4-6 unipolar-sheathed flagella that are continuous with the outer cell membrane (Wang et al., 2006). It is believed that the spiral shape of the bacteria and corkscrew movement provided by the sheathed flagella have enabled the organism to penetrate the viscous gastric mucus layer (Ottemann & Lowenthal, 2002). The ultra thin sections of H. pylori under electron microscopy also exhibited the typical cell wall structure of gram-negative bacterium that consists of both outer and inner membrane, with condensed cytoplasm containing nucleoid material and ribosome (Costa et al., 1999). On the other hand, the round shaped coccoid forms of H. pylori are more or less regarded as degenerative and dead cells (Kuster et al., 1997). There is another group of researchers who suggested that the coccoid form is viable but non-culturable (Hua & Ho, 1996; Saito et al., 2003). Substantial variation in cell wall structure, surface protein profiles and DNA contents were detected during the transition from spiral to coccoid (Benaissa et al., 1996; Costa et al., 1999), which hints that the two differential forms of H. pylori could contribute different roles during infection. Survey of Literature 2.1.3 Genetics of H. pylori The complete genome of H. pylori strain 26695 was reported by Tomb et al in 1997. It contains 1,667,867 base pairs of nucleotides and 1,590 predicted coding regions. A second complete genome of strain j99 was published in 1999 (Alm et al). Comparison of the two genomes revealed higher than 97% homology between them at the gene size and gene order with a limited number of discrete regions that are organized differently. When viewed in a genome wide manner, about 6-7% of the annotated genes are strain specific and are absent from each other with no homologue in the database (Alm et al., 1999). The prevalence of H. pylori infection varies in different geographical regions, ethnic backgrounds, socioeconomic conditions and age groups (Covacci et al., 1999), possibly contributed by the diversified bacterial genotypes of the isolates (Yamazaki et al., 2005). Unusual heterogeneity has been observed among the genotypes of clinical isolates and bacterial populations within the infected hosts, and the variation in bacterial population can be observed in individuals infected with more than one H. pylori strain (Blaser, 1990). The evolution of genotypic diversity in H. pylori may have been resulted from the presence of multiple strains within the same host, as plural cohabitation tends to favor the occurrence of free intraspecies recombination. 2.2 Virulence factors of H. pylori 2.2.1 Adhesins of H. pylori Many adhesins have been identified or predicted by the annotation of ORFs (open reading frames) in the H. pylori genome (Tomb et al., 1997; Alms et al., 1999). Most of them are outer membrane proteins that mediate receptor-ligand interaction between Survey of Literature bacteria and host escorting the adhesion of H. pylori onto the mucus layer (Doig et al., 1992). Blood group A antigen-binding adhesin (BabA) is one such adhesin that has been extensively studied. BabA is encoded by allelic babA2 gene and is involved in the binding of bacteria to the blood group antigen Lewis b surface epitopes of host (Boren et al., 1993; Ilver et al., 1998). Another newly identified adhesin is SabA (Sialic acidbinding adhesion) for binding to sialyl-Lewis x antigens in gastric epithelium in humans (Mahadavi et al., 2002). The adherence of H. pylori to sialylated glycoconjugates expressed during chronic inflammation contributes to the virulence and the extraordinary chronicity of H. pylori infection (Mahadavi et al., 2002). The other H. pylori proteins reported to be involved in the adherence of H. pylori onto the gastric epithelium are OipA (outer inflammatory protein, HP0638) (Yamaoka et al., 2002) and HopZ (homologue of porin) (Peck et al., 1999). It was noted that different numbers of CT dinucleotide repeats are characteristically present in the sequences of these adhesion genes (oipA, hopZ and sabA), which determine the functional status of these genes. In addition, it was also reported that the inclusion of the CT nucleotide repeats in the open reading frame would turn on the expression of these genes whereas the deletion and substitution of those repeats would cease the expression of these genes (Yamaoka et al., 2002). Interestingly, the functional status (on/off) of oipA, hopZ and sabA were found to affect the adherence and colonization properties of H. pylori (Yamaoka et al., 2002). Survey of Literature 2.2.2 Other pathogenic factors of H. pylori A number of H. pylori proteins have been established as virulence factors involved in its infection of the gastric mucosa. Among these factors, two major groups were classified as either related to adhesion and colonization of H. pylori or causing damage to host cells and benefiting the survival of bacteria in vivo. Among the bacterial proteins that have been reported to be crucial for the adhesion and colonization of H. pylori include flagellins that are responsible for bacterial motility (Josenhans et al., 1995; Ottemann & Lowenthal, 2002) and urease that neutralizes the acidic pH (Dunn et al., 1990; Tsuda et al., 1994; Karita et al., 1995). There are also various catalases and oxidases (Harris et al., 2003) that are enzymes leading to different biochemical degradations. Others include outer membrane proteins with or without known functions (Yamaoka et al., 2002), phospholipase (Dorrell et al., 1999) and numerous adhesins that mediate the adhesion of H. pylori to different host ligands. Mutations in the genes coding for these adhesive proteins in H. pylori have been found to reduce the adherence capability or colonization ability of the bacteria onto the gastric mucosa in animals (Evans et al., 2000). The other major group of virulence factors that causes tissue damage include vacuolating cytotoxinA (VacA) and cag pathogenicity island (cagPAI) which have been shown to be related to peptic ulcer and associated with the induction of immune response of the host (Le’Negrate et al., 2001). In addition, recent studies indicate that heat shock proteins e.g. HSP60 are necessary for the bacteria in combating against the hostile environment (Spohn et al., 2002). 10 References Ramphal R, Koo L, Ishimoto K S, et al. (1991). 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Luria-bertani (LB) Agar NaCl 10 g Yeast extract 5g Tryptone 10 g Agar 20 g Distilled water 1L The media was autoclaved at 121˚C for 15 minutes. The agar was then poured aseptically into sterile petri dishes and stored at 4˚C until use. (When required, Ampicillin stock solution was added to give a final concentration of 50 µg/ml.) 92 Appendi x Chocolate blood agar Blood Agar Base 20 g Horse blood 25 ml Distilled water 475 ml Blood Agar Base was mixed well and autoclaved at 121˚C for 15 minutes. The agar was cooled to 55˚C. A total of 25 ml horse blood was added to the agar aseptically and was then swirled in a 80˚C water bath for 10 minutes to lyse the blood cells. The chocolate agar was cooled to 55˚C before pouring into sterile petri dishes. Isopropyl-1-thio-D-Galactoside (IPTG) A stock solution of 100 mM of IPTG (Biorad) was prepared by dissolving in water. The solution was sterilized by filtering through 0.22 µm filter paper (Sartorious) and stored at –20˚C for use. Tris-EDTA (TE) buffer 10 mM Tris-HCl (Sigma) 1.576 g mM EDTA (Sigma) 0.372 g Distilled water 1L Adjust pH to 8.0 Lysozyme Lysozyme 100 mg Distilled water 10 ml 93 Appendi x Proteinase K Proteinase K 10 mg Distilled water ml RNAase Pancreatic RNAase 10 mg TE buffer ml 3M Sodium acetate Sodium acetate (Sigma) 61.5 g Distilled water 250 ml 50X Tris-acetate EDTA (TAE) buffer 40 mM Tris Base (Sigma) 4.85 g 0.114% (v/v) Glacial acetic acid (Merck) 57.1 ml mM EDTA (Sigma) 0.74 g Distilled water 1L qsp The working solution is 0.5 x for agarose gel electrophoresis. Ethidum bromide EtBr 100 mg Distilled water 10 ml 94 Appendi x EtBr was dissolved in the distilled water by stirring with a magnetic stirrer at room temperature and then stored in the dark at 4˚C. 6X Loading buffer 0.25% w/w Bromophenol blue 0.25 g 0.25% w/w Xylene cyanol 0.25g 30% v/v Glycerol 30 ml Distilled water 70 ml DNA Marker kb ladder marker (Gibco-BRL) HinDIII DNA marker ladder (New England Biolabs) 0.8% Agarose gel Agarose (Promega) 1.6 g 0.5 X TAE 200 ml 10 mg/ml EtBr 10 ul The 0.5X TAE buffer was added to the agarose and the mixture was heated in a microwave oven until all the agarose had dissolved. Ten µl of EtBr was added, and the mixture was mixed well and poured into a gel-casting tray. The agarose gel was left to solidify at room temperature. 95 Appendi x Buffers for His-tag affinity purification All the buffers used are prepared in stock solutions and diluted using Nanopure water. The final pH of the following solutions except for the charge buffer was adjusted to pH 7.9 at room temperature. 8X Binding buffer 40 mM Imidazole (Sigma) 0.68 g M NaCl 58.44 g 160 mM Tris-base 4.85g Nanopure water qsp 250 ml 8X Washing buffer 480 mM Imidazole 8.17 g M NaCl 58.44 g 160 mM Tris-base 4.85 g Nanopure water qsp 250 ml 8X Charging buffer 26.28 g 400 mM NiSO4 Nanopure water qsp 250 ml 4X Elute buffer 2.4 M Imidazole 40.85g 96 Appendi x M NaCl 14.61 g 80 mM Tris-base 2.42g Nanopure water qsp 250 ml 4X Strip buffer 400 mM EDTA 18.61 g M NaCl 14.61 g 80 mM Tris-base 2.42 g Nanopure water qsp 250 ml Buffers and reagents for SDS-PAGE Separating gel buffer Tris base Distilled water 18.15 g qsp 100 ml Fifty ml of distilled water was added to Tris base and concentrate HCl was used to adjust pH to 8.8. Distilled water was then added to make up a total volume of 100 ml. Stacking gel buffer Tris base Distilled water 12.1 g qsp 100 ml Fifty ml of distilled water was added to Tris base and concentrate HCl was used to adjust pH to 6.8. Distilled water was then added to make up a total volume of 100 ml. 97 Appendi x 10X Ammonium persulphate Ammonium persulphate 0.5 g Distilled water ml Dissolved and stored at –20˚C for use. 10% Sodium dodecyl sulphate (SDS) SDS 10 g Distilled water 100 ml 5X Sample loading buffer SDS 0.75 g M Tris HCl (pH 6.8) 1.563 ml b-Mercaptoethanol 1.25 ml Glycerol 2.5 ml Bromophenol blue 2.5 mg Sterile distilled water qsp 10 ml Electrophoresis buffer Tris base 3g Glycine 14.4 g SDS 1g Distilled water 1L 98 Appendi x Coomassie blue staining solution Coomassie blue R-250 2.0 g Methanol 400 ml Acetic acid 100 ml Distilled water 500 ml Destaining solution Methanol 400 ml Acetic acid 100 ml Distilled water 500 ml 4% Stacking gel 30% Acrylamide (Sigma) 670 ul M Tris-HCl (pH 6.8) 1.25 ml 10% SDS 50 ul 10% APS 25 ul Temed (Biorad) ul Distilled water qsp ml 10% Separating gel Acrylamide 3.3 ml 1.5 mM Tris- HCl (pH 8.8) 2.5 ml 10% SDS 100 ul 99 Appendi x 10% APS 50 ul Temed ul Distilled water qsp 10 ml Buffers for blotting Phosphate buffer saline (PBS) NaCl 8.0 g KCl 0.2 g Na2HPO4·H2O 1.15 g KH2PO4 0.2 g Distilled water 1L The final pH was adjusted to 7.6. Transfer buffer Tris base 5.82 g Glycine 2.93 g Methanol 200 ml Distilled water qsp 800 ml Substrate buffer 4-chloro-naphthol 0.06 g Cold Methanol 20 ml 100 Appendi x Substrate was dissolved completely in the dark before the addition of 100 ml PBS. Sixty µl Hydrogen peroxide was added immediately before use. Washing buffer (PBS-Tween20) Tween 20 0.5 ml PBS 1L Blocking solution Skim milk 2.5 mg PBS-Tween 20 50 ml Buffers and reagents for ELISA Carbonate buffer (pH 9.6) Na2CO3 1.59 g NaHCO3 2.93 g Distilled water 1L Serum diluent Tween 20 0.5 ml Thimerosal 0.2 g Gelatin (Dissolved by heating) 1g PBS (pH 7.6) 1L 101 Appendi x Conjugate diluent Bovine serum albumin (BSA) 0.02 g Thimerosal 0.2 g Gelatin 1g PBS (pH 7.6) 1L Phosphate-citrate buffer (Substrate buffer) Citric acid 2.5527 g Na2HPO4 4.5746 g Distilled water 500 ml The final pH was adjusted to pH 5.0 Stopping solution Concentrated H2SO4 Distilled water 34.06 ml qsp 250 ml Buffers for PAD assay Substrate buffer (1ml) 100 mM Benzoyl-Arg-ethyl-ester (BAEE) (Sigma) 1M Tris-HCl (pH 8.0) 50 µl 200 µl M Dithiothreitol (DTT) 10 µl 10 mM EDTA 100 µl 102 Appendi x 0.1mM Flavin mononucleotide (FMN) 10 µl Distilled water qsp ml Detecting reagent 0.5 % Diacetyl monoxime (Sigma) 0.5 g Distilled water 100 ml Stopping solution Concentrated H2SO4 24 ml Concentrated H3PO4 17 ml Distilled water 100 ml qsp 103 [...]... 3.5.3 Assay with mucins pretreated with neuraminidase and Na-metaperiodate In order to further characterize the nature of binding between rMBP protein and mucin, the biotin labeled mucins were pretreated with neuraminidase and Nametaperiodate, two commonly used agents for studying protein- lectin interactions by in vitro assays (Gentsch & Pacitti, 1985) Neuraminidase is also termed as Acetyl- 28 Materials... labeled mucin treated with neuraminidase before the reaction The pretreatment was carried out by incubating the biotin labeled mucin with neuraminidase (Sigma) overnight at 37°C with gentle shaking The pretreatment of biotin-labeled mucin with Na-metaperiodate was carried out by incubating the biotin labeled mucin with Na-metaperiodate (Sigma) dissolved in PBS overnight at 37°C with slow shaking 3.6 Detection... levels of affinity with mucins of various origins 2.6 Family of peptidyl-arginine deiminase (PAD) Peptidylarginine deiminase (PAD) is a family of enzymes that catalyze the conversion of protein- bound arginine to citrulline In mammalian cells, the enzyme is 15 Survey of Literature mainly involved in post-translational modification of proteins that could have a big impact on the structure and function... Pseudomonas aeruginosa outer-membrane proteins (OMPs) with heparin In the light of the effectiveness of this method, Western blot analysis was also employed for identifying the binding affinity of H pylori proteins with biotin-labeled mucins in this study 2.7.2 Solid phase carbohydrate binding assay Solid phase binding assay has served efficiently in identifying and screening the proteins of interest in many... difficile surface layer proteins (SLP) Mongodin et al (2002) investigated fibronectin binding protein (FnBPs) in Staphylococcus aureus by Western ligand affinity blotting analysis Downer et al (2002) used elastin as a ligand in Western blotting to identify elastin binding protein in S aureus (EbpS) By affinity chromatography and Western blotting assays, Plotkowski et al (2001) showed the interaction of... fibrillar hemagglutinin that recognizes sialic acid containing structures on erythrocytes and on mouse adrenal cells (Evans et al., 1988 & 1989) What has also been found is that mucins of different origin such as porcine gastric mucin, bovine submaxillary mucin and human salivary mucin are able to inhibit H pylori hemagglutination activity (Nakazawa et al., 1989; Piotrowski et al., 1991) These observations... Scientific Instruments) at 100V for 2 hours (Power Pac300, Biorad) Prestained precision marker (Biorad) was used as molecular weight marker After electrophoresis, SDS-PAGE gel was stained with Coomassie blue In brief, the gel was immersed in staining solution containing Coomassie Blue R-250 for 2 hours and then destained in destaining solution containing 10% acetic acid and 40% methanol with several changes... heated at 100°C for 25 minutes and the color development was optically measured at 492 nm The 1% PBS-BSA was used as the negative control The PAD assay was carried out over a pH range from 2.0 to 9.0 by adjusting the pH value of substrate buffer Meanwhile, the enzyme assay was tested on substrate solution containing free L-arginine instead of peptidyl-arginine Free L-arginine (Sigma) was dissolved in. .. glutaraldehyde in PBS (pH7.4) for 1 hour at 4˚C, and then washed three times with PBS for 5 minutes each Dehydration was done by incubating the cells in ascending graded series of ethanol at room temperature, including 5 min in 25%, 10 min in 50%, 20min in 75%, 20 min in 95% and 20 min in absolute ethanol for 3 changes Infiltration into LR White (Merck) was carried out by passing the sample through 1part... and Methods neuraminyl hydrolase (Sialidase) that catalyzes the hydrolysis of N-acetyl-neuraminic acid residues from glycoproteins and oligosaccharides (Gentsch & Pacitti, 1985) Nametaperiodate is a powerful oxidizing reagent catalyzing nonspecific oxidation of carbohydrate groups on various molecules such as glycoproteins and glycolipids (Marthieu et al., 1982) The assay was tested using biotin labeled . origin such as porcine gastric mucin, bovine submaxillary mucin and human salivary mucin are able to inhibit H. pylori hemagglutination activity (Nakazawa et al., 1989; Piotrowski et al.,. clinical isolates and bacterial populations within the infected hosts, and the variation in bacterial population can be observed in individuals infected with more than one H. pylori strain (Blaser,. most optimal for binding assay. The assay method has thus been established as a useful method for studying bacteria-mucin interaction in vitro and a large number of bacterial proteins have been