Human melanoma and Chinese hamster ovary cells galactosylate n-alkyl-{beta}-glucosides using UDP gal:GlcNAc {beta}1,4 galactosyltransferase

We previously showed that human melanoma, CHO and other cellscan convert ß-xylosides into structural analogs ofganglioside G_M3. We have investigated several potential acceptorsincluding a series of n-alkyl-ß-D-glucosides (n =6–9). All were labeled with ³H-galactose when incubatedwith human melanoma cells. Octyl-ß-D-glucoside (GlcßOctyl)was the best acceptor, whereas neither octyl--D-glucoside norN-octanoyl-methylglucamine (MEGA 8) were labeled. Analysis ofthe products by a combination of chromatographic methods andspecific enzyme digestions showed that the acceptors first receiveda single Galß1,4 residue followed by an 2,3 linkedsialic acid. Synthesis of these products did not affect cellviability, adherence, protein biosynthesis, or incorporationof radio-labeled precursors into glycoprotein, glycolipid orproteoglycans. To determine which ß1,4 galactosyltransferase synthesized Galß1,4GlcßOctyl,we analyzed similar incubations using CHO cells and a mutantCHO line (CHO 761) which lacks GAG-core specific ß1,4galactosyltransferase. The mutant cells showed the same levelof incorporation as the control, eliminating this enzyme asa candidate. Thermal inactivation kinetics using melanoma cellmicrosomes and rat liver Golgi to galactosylate GlcßOctylshowed the same half-life as UDP-Gal:GlcNAc ß1,4 galactosyltransferase,whereas LacCer synthase was inactivated at a much faster rate.We show that GlcßOctyl is a substrate for purifiedbovine milk UDP-Gal:GlcNAc ß1,4 galactosyltransferaseFurthermore, the galactosylation of GlcßOctyl by CHOcell microsomes can be competitively inhibited by GlcNAc orGlcNAcßMU . These results indicate that UDP-Gal:GlcNAcß1,4 galactosyltransferase is the enzyme used forthe synthesis of the alkyl lactosides when cells or rat liverGolgi are incubated with alkyl ß glucosides. alkylglucosides galactosyltransferase glycolipid artificial acceptors     

Pseudomonas aeruginosa Exploits Lipid A and Muropeptides Modification as a Strategy to Lower Innate Immunity during Cystic Fibrosis Lung Infection

Pseudomonas aeruginosa can establish life-long airways chronic infection in patients with cystic fibrosis (CF) with pathogenic variants distinguished from initially acquired strain. Here, we analysed chemical and biological activity of P. aeruginosa Pathogen-Associated Molecular Patterns (PAMPs) in clonal strains, including mucoid and non-mucoid phenotypes, isolated during a period of up to 7.5 years from a CF patient. Chemical structure by MS spectrometry defined lipopolysaccharide (LPS) lipid A and peptidoglycan (PGN) muropeptides with specific structural modifications temporally associated with CF lung infection. Gene sequence analysis revealed novel mutation in pagL, which supported lipid A changes. Both LPS and PGN had different potencies when activating host innate immunity via binding TLR4 and Nod1. Significantly higher NF-kB activation, IL-8 expression and production were detected in HEK293hTLR4/MD2-CD14 and HEK293hNod1 after stimulation with LPS and PGN respectively, purified from early P. aeruginosa strain as compared to late strains. Similar results were obtained in macrophages-like cells THP-1, epithelial cells of CF origin IB3-1 and their isogenic cells C38, corrected by insertion of cystic fibrosis transmembrane conductance regulator (CFTR). In murine model, altered LPS structure of P. aeruginosa late strains induces lower leukocyte recruitment in bronchoalveolar lavage and MIP-2, KC and IL-1β cytokine levels in lung homogenates when compared with early strain. Histopathological analysis of lung tissue sections confirmed differences between LPS from early and late P. aeruginosa. Finally, in this study for the first time we unveil how P. aeruginosa has evolved the capacity to evade immune system detection, thus promoting survival and establishing favourable conditions for chronic persistence. Our findings provide relevant information with respect to chronic infections in CF.     

Changes in the Activities of Various Glycosidases during Carrot Cell Elongation in a 2,4-D-Free Medium

Changes in the activities of some glycosidases were studiedin carrot suspension cultures with and without 2,4-D. Remarkablecell elongation occurs in a medium without 2,4-D, while fewcells elongate in a medium containing it. Glycosidases werefractionated into soluble, ionically wall-bound, tightly wall-boundand extracellular enzymes. The optimum pHs of all the ionicallybound glycosidases were in an acidic range, 4.4–5.0. The activities of the ionically and tightly bound ß-xylosidasesand ß-galactosidases were higher in elongating thanin non-elongating cells. Furthermore, the activities of theseenzymes increased with cell elongation during culture, suggestingthat they may play important roles in cell elongation. Higheractivities of soluble and cell wall-bound ß-glucosidaseand -mannosidase were found in non-elongating rather than inelongating cells. The activities of all soluble glycosidasesexcept ß-xylosidase were also higher in non-elongatingcells. Only ß-xylosidase and ß-galactosidaseactivities were detectable in the medium of the elongation culture. ¹ Present address: Department of Agricultural Chemistry, ObihiroUniversity of Agriculture and Veterinary Medicine, Obihiro,Hokkaido 080, Japan.     

Bovine recto-anal junction squamous epithelial (RSE) cell adhesion assay for studying Escherichia coli O157 adherence

Aim: To develop a new adherence assay, using cattle recto‐anal junction squamous epithelial (RSE) cells, for evaluating bacterial adherence to cells of bovine origin. Methods and Results: Proof of concept was demonstrated using the human gastrointestinal pathogen Escherichia coli O157:H7, for which cattle are reservoirs. Adherence assays were conducted using both RSE and HEp‐2 cells, in the presence and absence of D+Mannose. E. coli O157 specifically adhered in a type I fimbriae‐independent manner to RSE cells in significantly higher numbers and also bound significantly higher numbers of RSE cells than diverse laboratory strains of nonpathogenic E. coli. Conclusion: The RSE cell adhesion assay output highly reproducible and interpretable results that compared very well with those obtained using the more extensively used HEp‐2 cell adherence assay. Significance and Impact of the study: The RSE cell adhesion assay provides a convenient means of directly defining and evaluating pathogen factors operating at the bovine recto‐anal junction. The RSE cell adhesion assay further has the potential for extrapolation to diverse bacteria, including food‐borne pathogens that colonize cattle via adherence to this particular anatomical site.     

Glycoprotein Nature of Glycosidases from Leaves of Pisum sativum L

-Mannosidase, ß-N-acetylglucosaminidase, - and ß-galactosidaseand ß-glucosidase were partially purified from leavesof Pisum sativum by ammonium sulphate fractionation and columnchromatography on DEAE-Sephadex A-50 and hydroxylapatite. Atleast two molecular forms of each enzyme were resolved by thesetechniques except for ß-glucosidase of which onlyone form was resolved. Except for one form of -galactosidase,all of the glycosidases thus purified were completely boundby Sepharose-linked Concanavalin A. The binding was stronglyinhibited by cr-methyl-D-mannoside and no binding to Sepharose-6-Boccurred indicating that these glycosidases contain mannose-richoligosaccharides. The glycoprotein nature of -mannosidase, ß-galactosidaseand ß-glucosidase was further demonstrated by chromatographyon phenylboronate agarose columns. The differences in the concentrationof cr-methyl-D-mannoside and sorbitol required to elute thevarious glycosidases from Sepharose-linked Con A and phenylboronateagarose, respectively, suggested that these enzymes are glycosylatedto various degrees or that structural variation in their carbohydratemoieties occur. This is the first demonstration that glycosylationof several glycosidases present in a single plant species isapparently a generalized feature of these enzymes. Key words: Pisum sativum, Glycosidase, Glycoprotein     

In Vitro Evaluation of the Efficacy of a Silver-Coated Catheter

Bacteria commonly associated with nosocomial urinary tract infections were examined in vitro for their relative adherence to latex, 100% silicone-, hydrogel-coated latex-, and hydrogel/silver-coated latex urinary catheters. Degrees of adherence within 2 h were determined with cells radiolabeled with leucine. Adherence was greatest and equivalent on silicone and latex catheters. Adherence of four strains of Escherichia coli to the hydrogel/silver-coated catheter was decreased by 50% to 99% in comparison with the other catheters. Repeat testing with strains of E. coli and Pseudomonas aeruginosa with over 50 catheters demonstrated a consistency in the inhibition. The hydrophilic coating of the catheter appeared to be primary in the decreased adherence phenomenon followed by a secondary biocidal effect of the silver ion. Received: 2 December 1995 / Accepted: 3 January 1996     

Swimming Motility in a Longitudinal Collection of Clinical Isolates of Burkholderia cepacia Complex Bacteria from People with Cystic Fibrosis

Chronic bacterial lung infections in cystic fibrosis (CF) are the leading cause of morbidity and mortality. While a range of bacteria are known to be capable of establishing residence in the CF lung, only a small number have a clearly established link to deteriorating clinical status. The two bacteria with the clearest roles in CF lung disease are Pseudomonas aeruginosa and bacteria belonging to the Burkholderia cepacia complex (BCC). A number of common adaptations by P. aeruginosa strains to chronic lung infection in CF have been well described. Typically, initial isolates of P. aeruginosa are nonmucoid and display a range of putative virulence determinants. Upon establishment of chronic infection, subsequent isolates ultimately show a reduction in putative virulence determinants, including swimming motility, along with an acquisition of the mucoid phenotype and increased levels of antimicrobial resistance. Infections by BCC are marked by an unpredictable, but typically worse, clinical outcome. However, in contrast to P. aeruginosa infections in CF, studies describing adaptive changes in BCC bacterial phenotype during chronic lung infections are far more limited. To further enhance our understanding of chronic lung infections by BCC bacteria in CF, we assessed the swimming motility phenotype in 551 isolates of BCC bacteria from cystic fibrosis (CF) lung infections between 1981 and 2007. These data suggest that swimming motility is not typically lost by BCC during chronic infection, unlike as seen in P. aeruginosa infections. Furthermore, while we observed a statistically significant link between mucoidy and motility, we did not detect any link between motility phenotype and clinical outcome. These studies highlight the need for further work to understand the adaptive changes of BCC bacteria during chronic infection in the CF lung.     

Establishment of a Sensitized Immunoblotting Method for Measuring Plant Tubulin Content

A sensitized immunoblotting method was established for measuringsmall amounts of plant tubulin. The method involves electrophoretictransfer of protein including tubulin from SDS-polyacrylamidegels onto nitrocellulose paper, successive incubation of thenitrocellulose paper with a mouse monoclonal antibody to - orß-tubulin of chicken brain, an antibody to mouse IgGas the second antibody and the radioactive iodinated proteinA, and determination of the radioactivities of the bands onthe nitrocellulose paper thus probed. The radioactivities werelinearly proportional to the amounts of - or ß-tubulinfrom dark-grown Vigna mungo seedlings within a range of 4 to56 ng or of 4 to 32 ng, respectively. This method was used to estimate the tubulin contents of severalplant species using Vigna tubulin as a standard. -Tubulin contentsthus estimated were 25, 9, 19 and 11 µg-equivalents ofVigna tubulin per mg protein for Vigna seedlings, Daucus suspensioncells, Catharanthus suspension cells and Mougeolia cells, respectively.ß-Tubulin contents of Vigna, Daucus, Catharanthusand Mougeotia cells were 29, 10, 13 and 5 µg-equivalentsof Vigna tubulin per mg protein, respectively. (Received August 6, 1985; Accepted December 5, 1985)     

Adherence ability of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis to two different epithelial cell lines

The ability of 59 wild-type strains of Pseudomonas aeruginosa to adhere to the HeLa and Buffalo Green Monkey Kidney (BGMK) cells was investigated. Twenty strains were isolated from sputa of cystic fibrosis patients, while 19 strains were isolated from tracheal aspirates and 20 from bronchial secretions of patients without cystic fibrosis, and they were used as a control group of strains. The statistically significant difference between adherence ability of strains was observed (p < 0.01). While most of the tracheal and bronchial isolates were hyperadhesive (51-110 bacteria per cell) most of the cystic fibrosis isolates adhered poorly to the HeLa and BGMK cells (1-10 bacteria per cell). The bacterial binding to the cells was blocked when bacteria were incubated at 80 degrees C for 20 min before the adherence assay. These results indicate that alginate is not involved in the adherence of P. aeruginosa to the used epithelial cell lines, and, because of that, mucoid strains isolated from persistently colonized cystic fibrosis patients showed poor adherence ability.     

Pseudomonas aeruginosa inhibits endocytic recycling of CFTR in polarized human airway epithelial cells

The most common mutation in the CFTR gene in individuals with cystic fibrosis (CF), F508, leads to the absence of CFTR Cl^– channels in the apical plasma membrane, which in turn results in impairment of mucociliary clearance, the first line of defense against inhaled bacteria. Pseudomonas aeruginosa is particularly successful at colonizing and chronically infecting the lungs and is responsible for the majority of morbidity and mortality in patients with CF. Rescue of F508-CFTR by reduced temperature or chemical means reveals that the protein is at least partially functional as a Cl^– channel. Thus current research efforts have focused on identification of drugs that restore the presence of CFTR in the apical membrane to alleviate the symptoms of CF. Because little is known about the effects of P. aeruginosa on CFTR in the apical membrane, whether P. aeruginosa will affect the efficacy of new drugs designed to restore the plasma membrane expression of CFTR is unknown. Accordingly, the objective of the present study was to determine whether P. aeruginosa affects CFTR-mediated Cl^– secretion in polarized human airway epithelial cells. We report herein that a cell-free filtrate of P. aeruginosa reduced CFTR-mediated transepithelial Cl^– secretion by inhibiting the endocytic recycling of CFTR and thus the number of WT-CFTR and F508-CFTR Cl^– channels in the apical membrane in polarized human airway epithelial cells. These data suggest that chronic infection with P. aeruginosa may interfere with therapeutic strategies aimed at increasing the apical membrane expression of F508-CFTR. cystic fibrosis     

Carrier-Mediated ABA Uptake by Suspension-Cultured Phaseolus coccineus L. Cells: Stereospecificity and Inhibition by Ionones and ABA Esters

Astle, M. and Rubery, P. 1987. Carrier-mediated ABA uptake bysuspension-cultured Phaseolus coccineus L. cells: Stereospecificityand inhibition by ionones and ABA esters.—J. exp. Bot.38: 150–163. The substrate for the abscisic acid (ABA) carrier in Phaseoluscoccineus L. suspension-cultured cells is shown to be the (S)ABAenantiomer, K_m = 1?0 mmol m^–3. The methyl (MeABA) andphenyl (PheABA) esters of ABA inhibit carrier-mediated uptakeof ABA with half-maximal inhibition achieved at about 7?0 mmolm^–3 and 10 mmol m^–3 respectively: with (S)MeABAthis value is decreased to about 2?0 mmol m^–3. There isno demethylation of radioactive MeABA by the cells during 5min incubations. Although MeABA reversibly inhibits the ABAcarrier, it is not a transport substrate: association of radioactiveMeABA with living cells is unaffected by non-radioactive MeABAor ABA and, by comparison with frozen-and-thawed cells, it isshown that the radioactivity remains extracellular. It is proposedthat MeABA binds to the carrier to form an abortive complexthat is not translocated. The terpenoid ABA analogue LAB 144143also inhibits carrier-mediated ABA uptake. At concentrationsup to about 20 mmol m^–3 - and ß-ionone specificallyinhibit the ABA carrier with the half-maximal effect at about0?6 mmol m^–3 ß-ionone. However, at higher iononeconcentrations, the uptake of ABA, indol-3-yl acetic acid andof 5,5-dimethyloxazolidine-2,4-dione (DMO) are all stimulated:this may reflect general permeabilization of the membrane toweak acids by ionone. Key words: Uptake carrier, abscisic acid, methyl and phenyl esters of ABA, ionone, Phaseolus coccineus L. suspension culture     

Localization of ATPase Activity on the Plasmalemma of Scutellar Epithelial Cells of Germinating Barley (Hordeum vulgare L.)

Chaffey, N. J. and Harris, N. 1985. Localization of ATPase activityon the plasmalemma of scutellar epithelial cells of germinatingbarley (Hordeum vulgare L.).—J. exp. Bot 36: 1612–1619. ATPase activity has been localized at an ultrastructural levelin the absorptive region of the scutella of germinating barley(Hordeum vulgare L.). The enzyme is localized on the plasmalemmaof the epithelial cells. Using the Gomori reaction the depositionof reaction product on the plasmalemma, which is dependent uponthe presence of supplied ATP, was precluded or reduced by theinhibitors orthovanadate, mercuric chloride and DCCD, whilstß-glycerophosphate would not act as an alternativesubstrate. The mitochondria demonstrated phosphatase activitywith both ATP and ß-glycerophosphate as substrate.The results are discussed in relation to the active uptake ofmetabolites by the scutellum during germination and the structuralmodification of the plasmalemma of the epithelial cells to formplasmatubules. Key words: ATPase, Hordeum vulgare L., localization (ultrastructural)     

Monoclonal antibody LU-BCRU-G7 against a breast tumour-associated glycoprotein recognizes the disaccharide Gal{beta}1-3GlcNAc

The monoclonal antibody LU-BCRU-G7, that was generated by invitro immunization, shows clinical value as a prognostic markerin early stage breast carcinoma. It has now been characterizedwith regard to its binding epitope. Using a recently describedmethod based on the construction of N-substituted polyacrylamide(PAA) derivatives of carbohydrates (pseudopolysaccharides),the structure of the epitope for the monoclonal antibody LU-BCRU-G7has been determined. Competitive binding assays and inhibitoryenzyme-linked immunosorbent assays (ELISAs) using these pseudopolysaccharideshave shown the LU-BCRU-G7 epitope to be a disaccharide Galß1-3GlcNAc(Le^c; where Gal is D-galactose, Glc is D-glucose and GlcNAcis N-acetyl-D-glucosainine). Both galactose and N-acetyl glucosaminemoieties are essential for binding. Substitution on C-2 or C-3of the terminal galactose abolished binding, as did galactose-terminated oligosaccharides. The galactose moiety alone, asexpressed by the Galß-PAA conjugate, appeared to hea more important feature of the epitope than the GlcNAc-PAAconjugate, which failed to bind or inhibit the LU-BCRU-G7 antibody.In the N-acetyl glucosamine moiety, binding was decreased butnot eliminated by fucose substitution, as in Le^a, or changein configuration of C-4, as in Galß1-3GlcNAc. Omissionof the NAc group resulted in complete loss of activity. Thetetrasaccharide lacto-N-tetraose, although containing the terminalLe^c disaccharide, does not react with the antibody, suggestingconformational interference of the binding site. These findingsshow that the monoclonal antibody LU-BCRU-G7 recognizes a terminalisolactosamine fragment on a tumour-associated glycoprotein,which we have previously shown to be inversely related to survivalin breast cancer. breast cancer Galß1-3GlcNAc LU-BCRU-G7 monoclonal antibody pseudopolysaccharides     

Maternal Effects of mto1 Mutation, That Causes Overaccumulation of Soluble Methionine, on the Expression of a Soybean {beta}Conglycinin Gene Promoter-GUS Fusion in Transgenic Arabidopsis thaliana

ß-Conglycinin, the 7S seed storage protein of soybean(Glycine max [L.] Merr.), is comprised mainly of three subunits,designated , ' and ß. Expression of the gene encodingthe ß subunit is unique because its expression hasbeen shown to be down-regulated by exogenously applied L-methioninein immature soybean cotyledon cultures in vitro. Arabidopsisthaliana strain carrying a mto1-1 mutation overaccumulates solublemethionine. By using this mutant, we analyzed the effects ofmethionine on expression of the ß subunit gene invivo. Reciprocal crosses were made between the mto1-1 mutantand a transgenic A. thaliana strain, designated SNTß3,which carries a ß-glucuronidase (GUS) reporter geneunder the control of the promoter region of the ßsubunit gene. Analysis of GUS activity in F₁ seeds indicatedthat the GUS activity was dramatically repressed when the mto1-1mutant plants were used as female parents. We constructed astrain which carries both the transgene and mto1-1 mutationin the homozygous state. Analyses of the GUS activity in seedsof this double homozygous strain indicated that the GUS activitywas repressed to 2.5% of control by introduction of the mto1-1mutation. These results indicate that the ß subunitgene promoter activity in seeds is down-regulated by maternalgenotype and suggest that soluble methionine, or its mobilemetabolite, is translocated from mother plants to repress ßsubunit gene expression in seeds. ⁵Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan ⁶Present address: Department of Biotechnology, Faculty of Agriculture,The University of Tokyo, Bunkyo-ku, Tokyo, 113 Japan     

A 23 kDa membrane glycoprotein bearing NeuNAc{alpha}2-3Gal{beta}1-3GalNAc O-linked carbohydrate chains acts as a receptor for Streptococcus sanguis OMZ 9 on human buccal epithelial cells

Streptococcus sanguis colonizes several human oral surfaces,including both hard and soft tissues. Large salivary mucin likeglycoproteins bearing sialic acid residues are known to bindvarious S.sanguis strains. However, the molecular basis forthe adhesion of S.sanguis to human buccal epithelial cells (HBEC)has not been established. The present study shows that S.sanguisOMZ 9 binds to exfoliated HBEC in a sialic acid-sensitive manner.The desialylation of such cells invariably abolhhes adhesionof S.sanguis OMZ 9 to the cell surface. A soluble glycopeptidebearing short sialylated O-linked carbohydrate chains behavesas a potent inhibitor of the attachment of S.sanguis OMZ 9 toexfoliated HBEC. The resialylation of desialylated HBEC withCMP-sialic acid and Galß1,3GalNAc     

Adherence of Staphylococcus aureus to cell monolayers

Adherence of four strains of Staphylococcus aureus to eukaryotic cell monolayers was assayed with [3H]-thymidine labelled bacterial cells and the results were analysed by non-parametric statistical tests. Adherence to primary (human mesothelial) and semi-continuous (human embryonic lung) cell monolayers was significantly better than to continuous cell lines (HEp2, HeLa and Vero). HEp2 cell monolayers provided the most reliable assay substrate of the continuous cell lines tested. Variation occurred between bacterial culture batches but the assay measured significant differences between adhesion levels of the strains and distinguished between high level (RN92, 8325-4) and low level (Wood46, ISP458) adhering strains. Adherence to different batches of cell monolayers also varied but relative adherence values for strains were similar and the ranking of strains according to adhesion values was unchanged. Potential adhesion mediators have been monitored for their effect on adhesion of a highly adherent strain (RN92) to HEp2 monolayers. Fibronectin, protein A and anti-protein A did not significantly affect adhesion. Lipoteichoic acid caused a significant inhibition of adhesion. With critical statistical analysis to accommodate inherent variations, this assay provides a useful model to study factors involved in adherence of Staph. aureus to eukaryotic cells.     

Adherence ofStaphylococcus epidermidis to human pharyngeal epithelial cells: Evidence for lipase-sensitive adhesin and glycoprotein receptor

The adherence of eight strains ofStaphylococcus epidermidis to human pharyngeal epithelial cells (PEC) was investigated. All bacterial strains were nonencapsulated and non-slime producers. Binding was mannose resistant and was not related to surface hydrophobicity and surface charge of bacteria. Adherence to epithelial cells was reduced four- to tenfold (P<0.01) on pretreatment of bacteria with lipase while neuraminidase, phospholipase C, trypsin, and sodium periodate did not alter their binding. The surface carbohydrate profile of bacteria was studied by monitoring adherence to Lectin-Sepharoses. The bacteria did not conform to any pattern, and there was no relation to strain variation. The pretreatment of PEC with trypsin and sodium metaperiodate produced a marked reduction in bacterial binding (three- to 25-fold, P<0.01), but neuraminidase, phospholipase C, and lipase did not have any such effect. These findings provide evidence that the receptors on the surface of PEC are glycoprotein in nature, while the bacterial adhesin is a lipase-sensitive material.     

Glycosidases from Cotyledons of Pisum sativum L.

-Mannosidase and ß-N-acetylglucosaminidase were purifiedfrom extracts of cotyledons of germinating Pisum sativum L.A 13-fold purification of a-mannosidase free from ß-N-acetylglucosaminidaseactivity was achieved by precipitation in ammonium sulphate,column chromatography on DEAE-cellulose, and treatment with2 M pyridine. ß-N-Acetylglucosaminidase was purified200-fold by the use of (NH₄)₂SO₄, and chromatography on ConcanavalinA¹-Sepharose and Sephacryl-200. This preparation showed no measurablecontamination by -mannosidase activity. Both glycosidases appearto be glycoproteins and demonstrate optimal activity at pH valuesof 4.0–4.5. Both glycosidases appear to have very similarmolecular weights, with -mannosidase being slightly larger thanß-N-acetylglucosaminidase. An extensive search forthe activity of aspartylglycosylamine amido hydrolase in peacotyledons proved unsuccessful.     

Differential binding of Pseudomonas aeruginosa to normal and cystic fibrosis tracheobronchial mucins

Pseudomonas aeruginosa infection is a leading cause of deteriorationof pulmonary function in patients with cystic fibrosis (CF).The interaction of the bacterium with CF and non-CF tracheobronchialmucins was examined to understand the biochemical basis forthe high susceptibility of the lungs of CF patients to infectionby P.aeruginosa. The binding of radiolabelled bacteria to puremucins in solid-phase assays was not significantly above non-specificbinding to various blocking agents, such as bovine serum albumin,Tween 20, milk powder and polyvinyl pyrrolidine. Further, therewas a tendency for the bacteria to be excluded from plasticwells and membranes coated with mucin. Therefore, an indirectapproach involving the binding of radiolabelled P.aeruginosato asialo GM₁ ganglioside, the putative receptor for the bacteriaon tracheal cells, was used to compare the interaction of CFand non-CF mucins with the bacteria. Highly purified preparationsof CF mucin were consistently better inhibitors of the bindingof the bacteria to asialo GM₁ ganglioside than non-CF mucinpreparations. In the case of the binding of a stable mucoidstrain, the difference was statistically significant (P <0.001) at all concentrations of mucin tested. For the non-mucoidstrain, the difference was significant only at the higher concentrations.Of the saccharides tested similarly, sialyl lactose and theoligosaccharide portion of asialo GM₁ were found to be goodinhibitors. The increased binding of the bacteria to CF mucinwas further confirmed by a solution binding assay in which thebinding of ¹²⁵I-labelled mucin to unlabelled bacteria was determined.The binding of the bacteria to labelled CF and non-CF mucincould be inhibited by an excess of unlabelled human tracheobronchialmucin, but not by unrelated mucins, hyaluronic acid, alginicacid, bovine serum albumin and tetramethyl urea. The higherbinding of CF mucin, particularly to the mucoid strain of P.aeruginosa,is interesting and provides a model system to further investigatethe biochemical parameters of the interaction. asialo GM₁ ganglioside cystic fibrosis Pseudomonas aeruginosa respiratory mucins saccharide inhibitors.     

Glycoconjugates as possible receptors forStreptococcus pyogenes

The adherence capacities of M-protein-positive (M+) and M-protein-negative (M-) strains ofStreptococcus pyogenes were compared in human epithelial cells obtained from the pharynx (PEC) or from the buccal mucosa (BEC). Adherence to PEC was related to the presence of M protein (40.5±1.1 M+ and 17.8±0.6 M–S. pyogenes per PEC), whereas BEC showed adherence equally for M+ and M– strains. Different receptor sites may thus be involved on the two cell types. Preincubation of the bacteria with disialogangliosides (1 mg/ml), orN-acetylgalactosamine, ord-galactose (10 g/ml) resulted in diminished adherence of M+ strains to PEC but not to BEC. Chromatography ond-galactose-Sepharose 6B showed specific binding only of M+ group A streptococcal strains to gel beads. M– group A, and groups C and G streptococci did not bind. These observations suggest that the receptors on PEC for group A streptococci are distinct from those on BEC, and that most probably the attachment ofS. pyogenes to human pharyngeal cells occurs by specific, lectin-like binding to galactose residues on epithelial cells.