Biomedical subjects

F Ascencio

Publications and source records attributed to F Ascencio.

At least 37 records · Page 2Linked to original sources

PubMed1996

Immunochemical properties of a 60 kDa cell surface-associated heat shock-protein (Hsp60) from Helicobacter pylori.

Western blot analysis (immunoblotting) of cell surface-associated proteins from Helicobacter pylori confirmed our previous findings that binding of human IgG is a common property (among H. pylori strains). Purification of the IgG-binding proteins (IGBP) was achieved by two purification steps, affinity chromatography on IgG-Sepharose and nickel chelate affinity chromatography. SDS-PAGE and immunoblotting analysis revealed a 60 kDa protein with affinity for peroxidase labeled human IgG. Solid phase binding assays showed that IgG binds to an immobilized protein (IGBP). The 60 kDa IGBP binds human IgG1, IgG3 and IgM. Binding could be inhibited by the kappa chain of the human IgG, but not with its Fc fragment, nor with IgA or IgM. In addition, rabbit polyclonal antibodies raised against the 60 kDa IGBP blocked IgG binding. Monoclonal antibodies, specific to the Hsp60 heat shock protein of H. pylori recognized the 60 kDa IGBP as revealed by immunoblotting analysis, both in crude preparations and in the purified fractions.

Antibodies, Bacterial↗

PubMed1996

Cryptic domains of a 60 kDa heat shock protein of Helicobacter pylori bound to bovine lactoferrin.

Bovine lactoferrin binds to a 60 kDa heat shock protein of Helicobacter pylori. Binding ability was related to human immunoglobulin G because bovine lactoferrin binding proteins were isolated by extraction of cell surface associated proteins with distilled water, applied on IgG-Sepharose and nickel sulphate chelate affinity chromatography. Binding was demonstrated by Western blot after purified protein was digested with alpha-chymotrypsin and incubated with peroxidase-labeled bovine lactoferrin. Binding was inhibited by bovine lactoferrin, lactose, rhamnose, galactose, and two iron-containing proteins, ferritin and haptoglobin. Helicobacter pylori binds ferritin and haptoglobin via charge or hydrophobic interactions because this binding was not inhibited by specific and various glycoproteins or carbohydrates. Carbohydrate moieties of bovine lactoferrin molecules seem to be involved in binding because glycoproteins with similar carbohydrate structures strongly inhibited binding. Scatchard plot analysis of the binding of peroxidase-labeled bovine lactoferrin to H. pylori cells yielded a kd 2.88 x 10(-6) M. In addition, binding of H. pylori cells to bovine lactoferrin was enhanced when bacteria treated with pepsin or alpha-chymotrypsin after isolation from iron-restricted and iron-containing media.

Animals↗

PubMed1995

Cell-surface properties of the food- and water-borne pathogen Aeromonas hydrophila when stored in buffered saline solutions.

Aeromonas hydrophila, a ubiquitous inhabitant of aquatic environments, commonly expresses several cell-surface properties that may contribute to virulence. Since many aquatic microorganisms in hostile environments can withstand starvation conditions for long periods, we examined the effect of storage under nutrient-poor conditions on the expression of cell-surface properties of this pathogen. Phenotypes studied were: (1) cell-surface hydrophobicity and charge, and (2) the ability to bind connective-tissue proteins and lactoferrin. Our results suggest that the response of A. hydrophila to nutrient-poor conditions is regimen specific. Generally, A. hydrophila cells became more hydrophobic and significantly increased their ability to bind the iron-binding glycoprotein lactoferrin when the bacterium was stored under nutrient-poor conditions; however, under these conditions, the cells seemed to lose their ability to bind connective-tissue proteins.

Aeromonas hydrophila↗

PubMed1995

Cell-surface charge and cell-surface hydrophobicity of collagen-binding Aeromonas and Vibrio strains.

Partitioning in aqueous polymer two-phase systems of polyethylene glycol and dextran was used to detect and compare cell-surface charge and cell-surface hydrophobicity of Aeromonas hydrophila, A. caviae, A. sobria, Vibrio cholerae, and V. anguillarum strains. These strains have cell-surface components that bound either native or thermally denatured type I collagen (i.e., a mixture of the alpha1+alpha2 chains) and gelatin immobilized on latex beads. Our goals were: (1) to compare the possible relationship between the cell-surface charge/hydrophobicity and binding to collagen and (2) to evaluate the influence of the culture media on the expression of surface properties. There was no apparent relationship between cell-surface charge, cell-surface hydrophobicity, and binding to collagen. The expression of surface properties was dependent on the culture media. There was no relationship between binding to immobilized collagen and binding to soluble 125I-labeled collagen. Particle-agglutination reactivity differed when using various collagen-coated microbead preparations. There were general differences in the particle-agglutination reactivity when collagen-coated latex beads were prepared using different coating procedures. The negative charge and hydrophobicity of the various collagen-coated microbead preparations were also studied by partitioning in the two-phase system of polyethylene glycol and dextran. Under these conditions, the alpha1+alpha2 collagen-chain mixture covalently immobilized on carboxy-modified latex beads was less hydrophobic and negatively charged than gelatin and native collagen immobilized on the same kind of latex beads. For latex beads passively coated with collagen preparations, the alpha1+alpha2 collagen-chain mixture was more hydrophobic than gelatin and native collagen. We suggest that for screening collagen-binding among Vibrio and Aeromonas strains, a reliable and sensitive particle-agglutination assay should consider the collagen preparation and the coating procedure for the immobilization of collagen onto the latex beads. In this regard, carboxy-modified latex beads coated with an alpha1+alpha2 collagen-chain mixture gave the best results.

Aeromonas↗

PubMed1995

Helicobacter pylori interacts with heparin and heparin-dependent growth factors.

The pathogenic bacterium Helicobacter pylori, which causes active, chronic type B gastritis and peptic ulcer disease, and increases the risk for development of gastric cancer, could tentatively interfere with growth factors and growth factor receptors of importance for the gastroduodenal mucosa, e.g. heparin-binding FGFs (fibroblast growth factors). H. pylori binds FGF with an extremely strong affinity (3.8 x 10(-12)M), and also heparan sulfate and heparin with higher affinity (Kd 9 x 10(-9)M) than FGFs bind to heparin (10(-8) - 10(-9)M). FGF receptors are also dependent on heparin for their activation. Heparan sulfate binding proteins (HSBP) are exposed on and shed from the surface of H. pylori, which often are localised close to the epithelial stem cells in the gastroduodenal glands. H. pylori could thus efficiently interfere with growth factors and growth factor receptors, tentatively resulting in disturbance of the delicate balance that control the renewal, maintenance and repair of the gastroduodenal mucosa. This mode of action has previously not been considered, but may constitute part of its pathogenic mechanisms. Such a dynamic mode of action of H. pylori may explain the reason for that infected victims may either suffer from gastrointestinal symptoms or lack clinical evidence of disease or discomfort.

Fibroblast Growth Factor 1↗

PubMed1995

Particle agglutination assay for detection of albumin and IgG binding cell surface components of Helicobacter pylori.

Human serum albumin (HSA), bovine serum albumin (BSA) and human IgG were immobilized on latex beads to detect cell surface components of Helicobacter pylori binding to BSA, HSA, and IgG by a particle agglutination assay (PAA). In a total of 32 H. pylori strains tested, 16 strains interacted with BSA and 12 strains with HSA; 24 strains expressed binding of human IgG. The specificity of the agglutination reaction was studied by a particle agglutination inhibition assay performed by pre-incubating bacterial cell suspensions in buffers containing homologous proteins, unrelated glycoproteins and a number of common monosaccharides. Treatment of H. pylori cells with heat and proteolytic enzymes abolished binding to microbeads with immobilized IgG and albumin. IgG-binding surface components from cells of H. pylori strain CCUG 17875 were effectively extracted by incubating a cell suspension with 10 mM EDTA, 0.015 M sodium phosphate buffer (pH 7.2), and by washing H. pylori cells with distilled water. However, attempts to extract a fraction rich in albumin-binding components were unsuccessful. We conclude that cells of various H. pylori strains express commonly IgG-binding proteins, and that albumin-binding is probably mediated by non-specific hydrophobic interactions.

Antigens, Bacterial↗

PubMed1994

Coated-bead assay for microbial cell-associated and extracellular protease activity.

An assay utilizing latex beads coated with substrates of 125I-labelled connective tissue proteins is described as an alternative method for quantitating cell-associated and extracellular proteolytic activity of micro-organisms. Proteolytic activity is a function of the amount of 125I-labelled peptides released into the incubation mixture.

Candida albicans↗

PubMed1993

Binding of collagen, fibronectin, lactoferrin, laminin, vitronectin and heparan sulphate to Staphylococcus aureus strain V8 at various growth phases and under nutrient stress conditions.

We have examined how Staphylococcus aureus strain V8 cells interact with 125I-labelled extracellular matrix (ECM) and serum proteins (collagen type I and IV), fibronectin, lactoferrin, laminin, vitronectin, and heparan sulphate at various phases of the growth cycle. Maximal binding of these glycoproteins and heparan sulphate to the bacteria occurred after 17 to 20 h in the late stationary phase except for fibronectin-binding, which was maximal after 12 to 14 h. Binding of the glycoproteins and heparan sulphate to S. aureus V8 under nutrient stress conditions exhibited complex patterns based on different starving conditions and various binding ligands. In general, bacteria starved in distilled water and 0.02 M potassium phosphate buffer (pH 7.2) at room temperature showed high susceptibility to all binding ligands within the first 18 h, followed by entering a lower binding period (except for collagen-binding which still remained high). The binding was not correlated to cell surface charge or hydrophobicity of the bacteria. Furthermore, extracellular and cell-associated proteolytic activity of starved cells against ECM and serum proteins was found to be greater than for non-starved cells. Thus, S. aureus could sustain its ability to bind various connective tissue and cell surface components during a long period of time even in the absence of energy-yielding substrates.

Blood Proteins↗

PubMed1993

Studies on binding of glycosaminoglycans to Streptococcus pyogenes by using 125I-heparan sulphate as a probe.

Binding of 125I-heparan sulphate to the cell surface of Streptococcus pyogenes is mediated by proteins, that could be released from the streptococcal cell wall by using alkaline buffer. SDS-electrophoresis revealed two bands with molecular weights of 63 and 58 kDa. Binding of the 125I-labelled heparan sulphate probe to streptococci seems to be due to charge interactions, as the same probe was displaced by unlabelled heparan sulphate, other negatively charged molecules such as heparin, dextran sulphate, dermatan sulphate or by high ionic strength. The interaction was also strongly influenced by pH. The binding constant at pH 7.2 was estimated to be 9.8 x 10(6) mol/l, suggesting a moderate affinity. The presence of collagen of different types enhanced binding of 125I-labelled heparan sulphate to streptococci, whereas fibronectin and vitronectin had an inhibitory effect. The cooperation between heparan sulphate and collagen could be important for the adhesion of streptococci to connective tissue.

Bacterial Outer Membrane Proteins↗

PubMed1993

Isolation of a sialic acid-specific surface haemagglutinin of Helicobacter pylori strain NCTC 11637.

A deionized water extract of Helicobacter pylori NCTC 11637 contained haemagglutinin activity that was (i) soluble (i.e., not associated with particulate material sedimented by centrifugation at 100,000 x g for 1 h), (ii) stable to lyophilization, (iii) heat-labile, (iv) chymotrypsin-sensitive, (v) inhibited by fetuin, orosomucoid, and NANLac, but not by asialofetuin and (vi) inactive against guinea pig erythrocytes incubated with Clostridium perfringens neuraminidase, but active against untreated guinea pig erythrocytes. The data support the idea that the haemagglutinin is a protein which recognizes the alpha-(2-3) structure of sialylated glycoconjugates. Fractionation of the extract by isoelectric focusing and by gel filtration with Sephacryl S-400 indicated that the haemagglutinin has a pI of 3.7 and consist of high molecular-weight-protein aggregates. SDS-PAGE analysis of the preparation purified by gel filtration showed 3 protein bands at ca. 64 kD, 56 kD and 20 kD. Electron microscopy of H. pylori incubated with gold-labelled fetuin indicated that the haemagglutinin was associated with loosely adherent material on the bacterial surface, and that the purified haemagglutinin did not reveal a fimbrial structure. The ability to bind to sialoglycoconjugates on the erythrocyte membrane suggests that the haemagglutinin may be an important colonization factor enabling H. pylori to bind to similar saccharide structures on epithelial cells.

Animals↗

PubMed1993

Affinity of the gastric pathogen Helicobacter pylori for the N-sulphated glycosaminoglycan heparan sulphate.

Binding of 125I-heparan sulphate was a common property of Helicobacter pylori strains isolated from patients with gastroduodenal ulcer diseases. Binding was (i) saturable; (ii) reversible by the addition of unlabelled heparan sulphate and heparin; (iii) inhibited by unlabelled heparan sulphate, heparin, and heparin oligosaccharides but not by other glycosaminoglycans of comparable size (chondroitin sulphate and dermatan sulphate) or by highly glycosylated glycoproteins (hog gastric mucin and fetuin); (iv) reduced by heat treatment (80 degrees C, 10 min) and exposure of the bacteria to pronase E, proteinase K, trypsin and chymotrypsin, but unaffected by treatment with pepsin and neuraminidase; and (v) time-, pH-, and ionic strength-dependent. Scatchard plot analysis of the binding data indicated the presence of one class of high-affinity receptor (Kd = 9 x 10(-9) M) for heparan sulphate.

Binding, Competitive↗

PubMed1993

Rapid detection and characterization of sialic acid-specific lectins of Helicobacter pylori.

A particle agglutination assay (PAA) using fetuin (Ft) covalently coupled to carboxylate-modified latex (CML) particles was evaluated for rapid detection of sialic acid-specific haemagglutinins/lectins (SALs) of Helicobacter pylori isolates which bind sialoglycoconjugates. Sixty-three percent (20/32) of the isolates examined gave a positive PAA test. Cell-bound SALs were extracted by washing the bacteria with deionized water or isotonic saline, and their expression was influenced by pH and culture conditions. The Ft-CML reactivity of the PAA-positive isolates was inhibited by bovine submaxillary mucin, transferrin, fetuin, orosomucoid, vitronectin and lactoferrin in a manner which suggested that the isolates contain a lectin recognizing the alpha(2-6) linkage of terminal sialic acid. Western blots of strain NCTC 11637 SALs probed with horseradish peroxidase (HRP)-labelled Ft identified three bands (MW 64 kD, 62 kD, 56 kD) which also reacted with HRP-labelled mucin, transferrin, lactoferrin, orosomucoid, vitronectin and laminin. Sera from patients with a H. pylori infection and one polyclonal rabbit antiserum (strain NCTC 11637) also reacted with the SALs. Immunogold labelling of a polyclonal rabbit antiserum raised against the 64 kD protein of strain NCTC 11637 that reacted strongly with Ft-CML showed that abundant SALs were loosely cell-associated with the cell surface of both spiral and coccoidal forms of H. pylori. SALs were also present in low amounts on the surface of strain NCTC 11638 and 66, a clinical isolate that did not react with Ft-CML.

Animals↗

PubMed1993

The expression of potential colonization factors of yeasts isolated from fish during different growth conditions.

Three strains, Rhodotorula rubra, Rhodotorula glutinis, and Candida zeylanoides, isolated from fish, were tested for the expression of putative tissue colonization factors. All strains were able to bind collagen type I, fibronectin, and laminin to various degrees after growing on various solid and broth media, while the binding to collagen type IV was sparse under all conditions tested. For the three strains tested, a very low cell surface hydrophobicity was shown for growth on various solid and broth media. Mostly, the strains also expressed a negatively charged surface. Extracellular protease activity using different substrates was shown for all three strains. Furthermore, two properties related to iron scavenging, i.e., binding of lactoferrin and production of siderophores, were also tested. For the three strains a capacity to bind lactoferrin as well as a capacity to excrete siderophores were demonstrated. Since these different properties have been correlated to virulence and to the capacity of colonization in other organisms, we address the question of whether the expression of these properties in yeasts could contribute to colonization in fish.

Animals↗

PubMed1993

Expression of vitronectin and fibronectin binding by Candida albicans yeast cells.

Expression of binding to vitronectin (Vn or S-protein) and fibronectin (Fn) was common among clinical isolates of Candida albicans. Growth at 37 degrees C enhanced expression of both Vn and Fn binding. Some strains expressed higher binding after growth in liquid media and others after growth on solid media. Most strains expressed higher cell surface hydrophobicity after growth on agar media. Vn binding was less influenced by expression of cell surface hydrophobicity than Fn binding. Vn binding to yeast cells was optimal around pH 4 and Fn binding around pH 6. Binding to soluble Vn was inhibited by unlabelled Vn and to a lesser extent by Fn. Fn binding to the same C. albicans strain was inhibited by unlabelled Fn, Vn, fibrinogen and to some extent collagens. C. albicans strain 3248 expressed specific high binding of Vn, and high binding of Fn. Binding of both proteins was sensitive to heat and protease treatment, but in different ways. Vn binding differed significantly from the earlier reported Fn binding and may represent a novel type of tissue adherence.

Blood Proteins↗

PubMed1992

Surface properties, connective tissue protein binding and Shiga-like toxin production of Escherichia coli isolated from patients with ulcerative colitis.

Escherichia coli strains isolated from intestinal biopsies of patients with ulcerative colitis (n 146), Crohn's disease and colonic polyposis (n 41) were analysed for binding of collagen I, collagen IV, fibronectin and laminin. Strains expressed varying degrees of binding of one or more of the four connective tissue proteins. Only 32 strains did not express binding of any of the proteins. The strains expressed low or moderate cell surface hydrophobicity. There was no correlation between protein binding and expression of cell surface hydrophobicity. E. coli isolated from inflamed rectal mucosa were slightly less negatively charged than strains isolated from healthy intestinal mucosa. Shiga-like toxins I and II were detected in 32 strains from 28 patients. Of these, 5 strains had been isolated from normal or healed tissue. In patients with inflammatory bowel disease, connective tissue proteins are exposed in intestinal ulcerations. Strains expressing binding of one or several of these proteins may have a selective advantage to colonize these lesions.

Adolescent↗

PubMed1992

Vitronectin-binding surface proteins of Staphylococcus aureus.

S. aureus strain ISP 546 was selected (of 55 strains tested) to define optimal conditions for expression of vitronectin binding. High binding was expressed when the strain was grown on blood agar and in Todd-Hewitt broth. Binding was optimal in the 6.0 to 7.2 pH range and was unaffected by divalent cations and ionic strength. Binding was partially inhibited by D-mannose, heparin, types I and IV collagen, fibronectin, fibrinogen and vitronectin, but was not affected by other carbohydrates or glycoproteins tested. Cell surface binding components were extracted with the aid of 1 M LiCl (pH 5.0) from strain ISP 546 grown in Todd Hewitt broth. Vitronectin binding proteins were purified by affinity chromatography on heparin-Sepharose. Fractions inhibiting binding of 125I-labelled vitronectin to strain ISP 546 were eluted by 0.01 M NaOH, dialysed, concentrated and subjected to SDS-PAGE. Silver staining revealed one major band (70 kDa) and two minor bands (34 and 36 kDa).

Binding, Competitive↗

PubMed1992

Characterization of lactoferrin binding by Aeromonas hydrophila.

Various lactoferrin preparations (iron-saturated and iron-depleted human milk lactoferrins and bovine milk and colostrum lactoferrins) were bound by Aeromonas hydrophila. Binding was (i) reversible (65% of bound lactoferrin was displaced by unlabeled lactoferrin), (ii) specific (lactoferrin but not other iron-containing glycoproteins such as ferritin, transferrin, hemoglobin, and myoglobin inhibited binding), and (iii) significantly reduced by pepsin and neuraminidase treatment of the bacteria. The glycosidic domains of the lactoferrin molecule seem to be involved in binding since precursor monosaccharides of the lactoferrin oligosaccharides (mannose, fucose, and galactose) and glycoproteins which have homologous glycosidic moieties similar to those of the lactoferrin oligosaccharides (asialofetuin or fetuin) strongly inhibited lactoferrin binding. A. hydrophila also binds transferrin, ferritin, cytochrome c, hemin, and Congo red. However, binding of these iron-containing compounds seems to involve bacterial surface components different from those required for lactoferrin binding. Expression of lactoferrin binding by A. hydrophila was influenced by culture conditions. In addition, there was an inverse relationship between lactoferrin binding and siderophore production by the bacterium.

Aeromonas hydrophila↗

PubMed1992

Binding of heparan sulfate to Staphylococcus aureus.

Heparan sulfate binds to proteins present on the surface of Staphylococcus aureus cells. Binding of 125I-heparan sulfate to S. aureus was time dependent, saturable, and influenced by pH and ionic strength, and cell-bound 125I-heparan sulfate was displaced by unlabelled heparan sulfate or heparin. Other glycosaminoglycans of comparable size (chondroitin sulfate and dermatan sulfate), highly glycosylated glycoprotein (hog gastric mucin), and some anionic polysaccharides (dextran sulfate and RNA) inhibited heparan sulfate binding to various extents. Heat treatment (80 degrees C for 10 min) and treatment of the bacteria with pronase E, proteinase K, pepsin, and chymotrypsin considerably reduced their ability to bind 125I-heparan sulfate, but treatment with trypsin and neuraminidase did not affect binding. Scatchard plot analysis indicated the presence of cell surface components with low affinity (Kd = 3 x 10(-5) M) for heparan sulfate. Cell surface components were released by stirring bacteria with 1 M LiCl at 37 degrees C for 2 h. Proteins of this extract that competitively inhibited binding of 125I-heparan sulfate to S. aureus were isolated by affinity chromatography on heparin-Sepharose. Two proteins having molecular masses of approximately 66 and 60 kDa and the ability to bind 125I-heparan sulfate were obtained. The first 9 amino-terminal amino acid residues of the 66-kDa protein are Asp-Trp-Thr-Gly-Trp-Leu-Ala-Ala-Ala, and the first 4 amino-terminal amino acid residues of the 60-kDa protein are Met-Leu-Val-Thr.

Amino Acid Sequence↗