Biomedical subjects

D L Hasty

Publications and source records attributed to D L Hasty.

At least 55 records · Page 3Linked to original sources

PubMed1990

Colonial morphology of staphylococci on Memphis agar: phase variation of slime production, resistance to beta-lactam antibiotics, and virulence.

The growth of Staphylococcus epidermidis sensu stricto and Staphylococcus saprophyticus on Memphis agar yielded up to 6 morphotypes with each strain. With S. epidermidis, one morphotype produced slime (rho) but became non-slime-producing (epsilon) at a high frequency. The slime-producing rho variants were methicillin-resistant and more virulent than methicillin-susceptible epsilon variants in an endocarditis model. With S. saprophyticus, phase variation was of higher frequency. Nitrosoguanidine mutagenesis produced a stable blue epsilon form that was more virulent than the parent in a mouse model of urinary tract infection. Mutants with the blue epsilon phenotype differed from gold epsilon parents in a variety of phenotypic properties, including increased resistance to oxacillin. These staphylococcal species have a high frequency of phase variation: Phase variants differ in antibiotic resistance and virulence, which is only partially correlated with suggested virulence factors such as slime production.

Animals↗

PubMed1988

Adherence of Streptococcus sanguis to conformationally specific determinants in fibronectin.

The adherence of Streptococcus sanguis to specific receptors exposed or deposited at the site of endothelial damage may play an important role in the development of infective endocarditis. Adherence of the Challis strain of S. sanguis to gelatin (or collagen) and gelatin-binding components of plasma was examined with an enzyme-linked immunosorbent assay. S. sanguis adhered poorly to immobilized gelatin and to molecular or fibrillar collagen. However, in the presence of fresh human plasma, the adherence of S. sanguis to all three substrates increased as much as eightfold. Removal of gelatin-binding proteins eliminates the ability of plasma to enhance adherence of S. sanguis to the substrates. Addition of purified human plasma fibronectin (Fn) to the absorbed plasma restored the adherence-promoting ability in a dose-dependent manner. A similar dose-dependent increase in S. sanguis adherence was observed when increasing concentrations of Fn alone were added to the gelatin-coated assay wells. S. sanguis adherence to immobilized fibronectin could not be inhibited by preincubating either the bacteria or the gelatin-coated assay wells with Fn or by including excess soluble Fn in the assay mixture. Studies with peptides purified from trypsin digests of Fn indicated that the 160- to 180-kilodalton (kDa) fragments which retain both the gelatin-binding and the cell-binding regions of the intact molecule support adherence of S. sanguis to gelatin. The 160- to 180-kDa fragments inhibited the interaction of S. sanguis with immobilized Fn. In contrast, intact Fn and the 31-kDa amino-terminal fragment were unable to inhibit the adherence when used in equivalent or greater molar amounts. These in vitro results suggest that in the presence of whole plasma, S. sanguis binds to immobilized gelatin or collagen via Fn bound to the immobilized substrates. Our finding that adherence of S. sanguis to immobilized Fn can occur in the presence of large concentrations of Fn, whether in plasma or purified, indicates that a S. sanguis-binding domain is cryptic in the Fn molecule while in solution and is exposed by a conformational change when the Fn becomes bound to gelatin-coated plastic. The ability of peptide fragments of Fn to inhibit S. sanguis adherence is consistent with this hypothesis.

Bacterial Adhesion↗

PubMed1987

Hybridoma antibodies to the lipid-binding site(s) in the amino-terminal region of fibronectin inhibits binding of streptococcal lipoteichoic acid.

In this report, we present evidence to suggest that streptococci and lipoteichoic acid (LTA) interact with a fatty acid binding site located near the NH2-terminus of fibronectin. The evidence is based on the following observations. Antibodies directed against a synthetic peptide (residues 1-30 of the amino-terminus of fibronectin) reacted with the two thermolysin-generated peptides (24 and 28 kilodaltons [kDa]) that were adsorbed by and eluted from streptococci. The adsorption of the 24- and 28-kDa peptides to streptococci was inhibited by LTA. The two monoclonal antibodies that inhibited the binding of LTA to fibronectin reacted only with the 24- and 28-kDa fragments of fibronectin. Conversely, LTA, as well as lauric acid and oleic acid, blocked the binding of the same monoclonal antibodies to fibronectin. LTA had no effect on the binding of hybridoma antibodies directed against the collagen or cell-binding domain.

Animals↗

PubMed1987

Effects of fibronectin and other salivary macromolecules on the adherence of Escherichia coli to buccal epithelial cells.

The effect of saliva and fibronectin (Fn) on the adherence of a type 1 fimbriated strain of Escherichia coli to human buccal epithelial cells was studied. Saliva pretreatment of epithelial cells led to a dose-dependent increase in adherence that was inhibited by alpha-methyl mannoside, which is typical of a type 1 fimbria-mediated event. The molecules responsible for affecting this increased adherence were nondialyzable and were recovered after lyophilization. E. coli adherence was stimulated by individual saliva samples from each of 11 volunteers. Fn inhibited E. coli adherence to saliva-treated buccal cells by more than 60%. Biotinylated E. coli and Fn were reacted with Western blots of whole saliva to identify the receptors that might explain the phenomenon described above. Both E. coli and Fn bound to 57- and 62-kilodalton (kDa) protein bands in Western blots of sodium dodecyl sulfate gels of whole saliva. The binding of E. coli to these bands was inhibited by pretreatment with unlabeled Fn. To study these salivary components, samples of saliva were electrophoresed on sodium dodecyl sulfate gels, strips corresponding to the appropriate molecular weights were cut out, and the proteins were eluted electrophoretically. Material that eluted from strips at 57 and 62 kDa, but not that from a control strip, stimulated E. coli adherence to buccal cells. Alternatively, saliva was fractionated over 100- and 50-kDa cutoff filters. Of the three fractions obtained, only the fraction passing through the 100-kDa filter and retained by the 50-kDa filter stimulated E. coli adherence to buccal cells. This fraction also increased the binding of Fn to buccal cells. These observations suggest the possibility that one or more salivary components bind to the surface of buccal cells and serve as receptors for type 1 fimbriated E. coli. Fn also binds to this isolated material; and it is apparently by these interactions, at least in part, that saliva stimulates and Fn inhibits E. coli adherence. The way in which these interactions may affect bacterial adherence in vivo remains to be elucidated.

Bacterial Adhesion↗

PubMed1986

Isolation and characterization of a 60-kilodalton salivary glycoprotein with agglutinating activity against strains of Streptococcus mutans.

A bacterial agglutinin specific for strains of Streptococcus mutans was isolated from human saliva. Physiochemical analyses showed the agglutinin to be a glycoprotein with a molecular weight of 60,000. The agglutinin aggregated four of the eight strains of Streptococcus mutans tested but did not aggregate the strains of Streptococcus salivarius, Streptococcus sanguis, and Streptococcus mitis tested. Chemical modification of carbohydrate moieties of the agglutinin with sodium metaperiodate had no effect on aggregation, whereas modification of the polypeptide portion with trypsin abolished aggregating activity. A set of five murine hybridoma antibodies was employed to further analyze the agglutinin. Two carbohydrate-specific antibodies, directed against D-mannose and N-acetylgalactosamine moieties, respectively, failed to block agglutinin- or whole saliva-mediated aggregation of S. mutans cells. In contrast, two antibodies directed against pronase-sensitive antigenic sites blocked both agglutinin- and saliva-mediated aggregation of S. mutans cells. Western blot analysis with the agglutinin-specific hybridoma antibodies demonstrated the agglutinin in whole saliva and in artificial tooth pellicles formed on hydroxyapatite beads incubated with saliva. These results suggest that a 60-kilodalton glycoprotein of human saliva is a bacterial agglutinin with specificity for certain strains of S. mutans. They further suggest that aggregation is mediated by polypeptide rather than carbohydrate determinants of the glycoprotein.

Agglutination↗

PubMed1986

Binding of Streptococcus pyogenes to soluble and insoluble fibronectin.

The interaction of soluble and insoluble fibronectin with Streptococcus pyogenes was investigated. Soluble fibronectin bound to S. pyogenes in a dose-dependent and irreversible manner. Lipoteichoic acid competitively inhibited the binding of fibronectin to S. pyogenes but had little effect on the binding of fibronectin to staphylococci or pneumococci. The phase of growth of the streptococci had a slight effect on binding of fibronectin, with optimal binding occurring in the late log phase. S. pyogenes cells bound to fibronectin immobilized on microtiter plates in a dose-dependent and saturable manner. Both soluble fibronectin and lipoteichoic acid inhibited the binding of streptococci to immobilized fibronectin, suggesting that streptococci interact with soluble and insoluble fibronectin in a similar manner. Antibodies to fibronectin blocked the attachment of streptococci to immobilized fibronectin, whereas normal serum had no effect. Adherence of streptococci to buccal epithelial cells was inhibited by antibodies to fibronectin, but not by normal sera or by antibodies to buccal epithelial cells. The data suggest that lipoteichoic acid on the surface of S. pyogenes binds to fibronectin exposed on the host cell and that such binding mediates the attachment of streptococci to host cells.

Adhesiveness↗

PubMed1986

Immunochemical and ultrastructural mapping of the gelatin-binding and cell-attachment regions of human plasma fibronectin with monoclonal antibodies.

Monoclonal antibodies against fibronectin were used to locate the gelatin-binding and cell-attachment regions of plasma fibronectin at an ultrastructural level. A total of 23 hybridomas were generated using mice immunized with either intact fibronectin or a 40 000 Mr gelatin-binding fibronectin fragment. One of these antibodies (D9b) strongly inhibited the interaction of radio-labelled fibronectin with gelatin. Another antibody (IB10) inhibited the attachment of Chinese hamster ovary (CHO) cells to a fibronectin substratum by 99%. Both of these antibodies were purified by affinity chromatography on columns of fibronectin-Sepharose and were then incubated with soluble fibronectin to form antigen-antibody complexes. The complexes were separated from free antibody on a column of Sephadex G-200 and were prepared for electron-microscopic examination by spraying on mica discs and rotary shadowing with platinum. As determined by this method, the fibronectin molecules measured 124 +/- 1.7 nm in length. Monoclonal antibody IB10 was visualized as a globular projection 40 +/- 1.4 nm from one end of the fibronectin filament. Monoclonal antibody D9b, on the other hand, was visualized as a globular projection at or near one or both ends of the molecule. These data provide the first morphological localization of the gelatin-binding and cell-attachment regions of fibronectin and indicate that further studies using monoclonal antibodies directed toward other epitopes should shed light not only on function but also on the tertiary and quaternary structure of the fibronectin molecule.

Animals↗

PubMed1985

Physicochemical and immunological studies of the renatured alpha 1(II) chains and isolated cyanogen bromide peptides of type II collagen.

The alpha 1(II) and cyanogen bromide (CB)1-generated peptides of chick type II collagen were isolated, purified, renatured and examined for their physicochemical and immunological properties. The alpha 1(II) chains and peptides CB-6 through CB-12 (3,000 to 40,000 daltons) formed renatured thermostable products as determined by measurements of reduced viscosity, optical rotation and Stokes radius. Moreover, renatured alpha 1 (II) chains and CB-10 were observed to form segment-long-spacing (SLS) crystallites under appropriate conditions. When examined for immunoreactivity with defined rat polyclonal and mouse monoclonal antibodies to chick type II collagen, conformation-dependent epitopes were detected on renatured alpha 1(II) chains and renatured peptides, CB-8, CB-10 and CB-11. Conformation-independent epitopes were also detected on all CB-peptides in their denatured form. These studies demonstrate that the alpha 1 (II) chains and CB-peptides of chick type II collagen can be efficiently renatured and that the renatured products retain some conformation-dependent epitopes present on the naive molecule.

Animals↗

PubMed1985

Location of a fibronectin domain involved in newt epidermal cell migration.

The interaction of migrating newt epidermal cells with the extracellular matrix protein, fibronectin, was studied. Pieces of nitrocellulose coated with intact human plasma fibronectin or proteolytically derived fragments were implanted into wounded limbs so that the coated nitrocellulose served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Epidermal cells migrated very poorly on nitrocellulose pieces coated with (a) a 27-kD amino-terminal heparin-binding fragment, (b) a 46-kD gelatin-binding fragment, (c) a combined 33- and 66-kD carboxy-terminal heparin-binding preparation representing peptide sequences in the A and B chains, respectively, or (d) a 31-kD carboxy-terminal fragment from the A chain, containing a free sulfhydryl group. In contrast, epidermal cells readily migrated onto nitrocellulose coated with a mixture of fragments from the middle of the molecule (80-125kD) that bind neither heparin nor gelatin. Attempts to block migration on fibronectin-coated nitrocellulose using IB10, a monoclonal antibody that blocks Chinese hamster ovary cell attachment to fibronectin, were unsuccessful despite saturation of the epitope against which IB10 is directed. In contrast, a polyclonal anti-fibronectin antibody did inhibit migration. These results show that the ability of fibronectin to support newt epidermal cell migration is not shared equally by all regions of the molecule, but is restricted to a domain in the middle third. They also suggest that the site supporting migration is separate and distinct from the site mediating Chinese hamster ovary cell attachment.

Animals↗

PubMed1985

Binding of fibronectin to human buccal epithelial cells inhibits the binding of type 1 fimbriated Escherichia coli.

The interaction of purified human plasma fibronectin (FN) with human buccal epithelial cells was studied. Maximal binding of FN occurred at pH 5. The majority of the binding was specific and reversible. The binding of FN to buccal cells was saturable, reaching a maximum when 10(5) buccal cells were incubated with approximately 200 micrograms of radiolabeled protein per ml. The adherence of a type 1 fimbriated strain of Escherichia coli to buccal epithelial cells was inhibited by the addition of FN in a dose-related manner. Our results indicate that exogenous FN can bind to human buccal epithelial cells and block the attachment of a type 1 fimbriated strain of E. coli.

Adhesiveness↗

PubMed1985

Protection against Escherichia coli-induced urinary tract infections with hybridoma antibodies directed against type 1 fimbriae or complementary D-mannose receptors.

Hybridoma antibodies directed against quaternary structural epitopes of the type 1 fimbrial adhesin of Escherichia coli or against D-mannose, the sugar determinant in the complementary host cell receptor, prevented the attachment of mannose-sensitive E. coli to various eucaryotic cells. Passive intraperitoneal administration of the fimbria-specific or D-mannose-specific antibodies protected mice against retrograde colonization with mannose-sensitive E. coli instilled into their urinary bladders. Monoclonal antibodies directed against fimbrial subunits rather than quaternary structural epitopes or against N-acetylgalactosamine rather than D-mannose residues lacked protective activity. These studies provide evidence that bacterial colonization can be blocked or interrupted by antibodies directed against either the adhesin or the complementary host cell receptor of pathogenic microorganisms.

Adhesiveness↗

PubMed1983

Repeating covalent structure and protective immunogenicity of native and synthetic polypeptide fragments of type 24 streptococcal M protein. Mapping of protective and nonprotective epitopes with monoclonal antibodies.

The complete amino acid sequences of three cyanogen bromide peptide fragments (CB3, CB4, and CB50 of type 24 M protein extracted from Streptococcus pyogenes by limited pepsin digestion were determined by automated Edman degradation of the uncleaved peptides and their tryptic peptides. CB3 and CB4 each contain 35 amino acid residues, whereas CB5 contains 37. The sequence of CB3 was found to be: (formula: see text) (where Hse represents homoserine). The sequence of CB4 was identical except for amino acid substitutions of arginine and glutamine at positions 23 and 24, respectively. The sequence of CB5 also was identical with that of CB3 except for substitutions of aspartic acids at positions 28 and 29; leucine, glutamic acid, and glycine at positions 33, 34, and 35, respectively; and an additional two amino acids, alanine and homoserine, at positions 36 and 37, respectively. A comparison of the structures of these three peptide fragments with those previously reported for CB6 and CB7 revealed as few as one to six amino acid substitutions among the five repeating peptides; CB4 and CB6 differed only by a single Asp/Glu substitution at position 26. When covalently linked to polylysine and injected as an emulsion in complete Freund's adjuvant, CB3, CB4, and CB5 each evoked high titers of type-specific opsonic and bactericidal antibodies in rabbits. A chemically synthesized peptide identical with native CB3 except that it contained methionine instead of homoserine at its COOH terminus was similarly immunogenic. None of the conjugated native or synthetic peptides raised antibodies at reacted in immunofluorescence tests with sarcolemmal membranes of human heart tissue. Mapping studies with monoclonal antibodies revealed a number of distinct protective and nonprotective epitopes. The single Asp/Glu substitution between CB4 and CB4 rendered the 35-residue peptide unrecognizable by protective monoclonal antibodies but recognizable by a nonprotective one. Our studies demonstrate that the repeating covalent structures of native and chemically synthesized polypeptide fragments of streptococcal M protein possess several unique as well as repeating epitopes that evoke opsonic and presumably protective, but not heart cross-reactive, antibodies against a rheumatogenic strain of S. pyogenes.

Amino Acid Sequence↗

PubMed1983

Antiadhesive properties of a quaternary structure-specific hybridoma antibody against type 1 fimbriae of Escherichia coli.

The relationship between the structure and biological function of type 1 fimbriae of Escherichia coli was investigated using a set of monoclonal antibodies directed against conformation-specific antigenic determinants. Of three monoclonal antibodies tested, only one (clone CD3) prevented adhesion of the vaccine strain to epithelial cells or guinea pig erythrocytes. The antibody produced by CD3, but not that produced by the other two hybridoma clones (AA8 and GG1), precipitated isolated fimbriae by double diffusion in agar gel and was shown to bind in a highly discrete, periodic manner along the length of each of the fimbriae by immunoelectron microscopy. Immunoelectroblots of type 1 fimbrial subunits and polymers electrophoresed in SDS-gels indicated that the antibodies in AA8 and GG1 reacted only with fimbrial monomers (mol wt 17,000), whereas the antibody in CD3 reacted only with polymers of mol wt 102,000 (hexamers) or higher. ELISA inhibition assays demonstrated that dissociated fimbrial subunits lost their reactivity with antibody CD3 but gained reactivity with antibodies AA8 and GG1. Conversely, when allowed to reassemble in vitro in the presence of 5 mM MgCl2, the reassembled fimbriae lost their reactivity with antibodies AA8 and GG1 but regained reactivity with antibody CD3. These results demonstrated that certain antigenic epitopes are dependent on quaternary structural determinants, whereas others are independent of quaternary fimbrial structure and also are inaccessible for antibody binding in fimbriae once they have been assembled. These monoclonal antibodies should prove useful in studies of the structural determinants of the biological function of type 1 fimbriae as well as in studies of fimbrial synthesis, transport, and assembly.

Adhesiveness↗

PubMed1983

Freeze-fracture studies of neonatal mouse incisors.

Differentiation of preodontoblasts to odontoblasts and preameloblasts to ameloblasts during development of the mouse mandibular incisor proceeds in a gradient from the area of the odontogenic organ, where undifferentiated ectomesenchymal and epithelial cells proliferate, toward the incisal tip where mature tooth tissues, dentin and enamel, are present. The freeze-fracture technique has been used in the work presented here to study cell membrane ultrastructure of preodontoblasts and preameloblasts at several stages of differentiation. At early stages of differentiation, cuboidal preameloblasts are joined together distally by numerous gap junctions. Relatively fewer junctions occur elsewhere on the lateral plasma membranes, but gap junctions frequently occur proximally between preameloblasts and stratum intermedium cells. As differentiation proceeds and the cells become columnar, distal and proximal junctions persist. Tight junctions, however, were not observed at any of the stages studied. Intramembrane particle concentration of the lateral preameloblast plasmalemma appears to increase as differentiation proceeds. Odontoblasts are also joined distally by numerous gap junctions which persists through later stages of differentiation. Although odontoblast cell processes were observed to project toward the preameloblast layer, no clear points of cell to cell contact or defined intercellular junctions between the two cell types were observed.

Ameloblasts↗

PubMed1983

Immunocytochemical localization of fibronectin in human fibroblast cultures using a cell surface replica technique.

The pericellular fibronectin-containing matrices of human foreskin fibroblasts cultured in ascorbate-supplemented medium were examined using surface replicas. An extensive filamentous network is present over and between adjacent cells, with a considerable amount at points of cell-to-cell contact. Indirect immunocytochemical localization of the distribution of fibronectin and procollagen type III within the matrix was done using the peroxidase-antiperoxidase (PAP) sandwich technique. The PAP molecule with the surrounding diaminobenzidine reaction product appears as a globular particle of approximately 39 nm in surface replicas. The apparent size of the marker was larger (60-80 nm) when bound to pericellular fibronectin, due presumably to the binding of more than one PAP complex to each fibronectin molecule. The immunocytochemical data suggest that fibronectin is a component of most, if not all, matrix fibrils. Some of the smallest filaments of the matrix (5-10 nm) exhibit a periodic, beaded appearance, with a repeat distance of approximately 70-100 nm. After either anti-fibronectin or anti-procollagen type III labeling, the filaments were decorated at regular 70-100 nm intervals with the globular marker. We suggest that the periodicity may be due to fibronectin molecules bound to collagen microfibrils at regular intervals. Our results demonstrate the usefulness of combined surface replica and immunocytochemical techniques for analysis of matrix components of cultured cells.

Cells, Cultured↗

PubMed1982

Production and preliminary characterization of monoclonal antibodies to rat plasma fibronectin.

We have produced several monoclonal antibodies which appear to be directed against different antigenic determinants of rat plasma fibronectin. Fibronectin was purified from rat plasma by affinity chromatography on gelatin-Sepharose and arginine-Sepharose columns. Mice were immunized and hybridomas were prepared by fusing spleen cells with Sp2/0-Ag14 myeloma cells using poly(ethylene glycol). Three hybridomas (RFN1, RFN2 and RFN3) were selected for characterization. All are IgG molecules, one is IgG2a, one IgG2b and one IgG1. Titers of ascites fluids produced using these hybridomas range from 102 400 to greater than 409 600. The antibodies cross-reacted to different degrees with human fibronectin. Rat fibronectin was radioactively labeled and cleaved using human polymorphonuclear leukocyte elastase. Four major peptides, Mr approx. 160 000, 140 000, 60 000 and 30 000 were produced. Each of the hybridoma antibodies immunoprecipitated different elastase peptides. RFN1 precipitated the Mr 160 000 peptide, RFN2 precipitated the Mr 160 000 and the Mr 140 000 peptide and RFN3 precipitated the Mr 60 000 peptide as well as low molecular weight material migrating at the buffer front. These antibodies will be useful in studies of structure/function relationships of rat fibronectin.

Animals↗

PubMed1982

Hybridoma antibodies against protective and nonprotective antigenic determinants of a structurally defined polypeptide fragment of streptococcal M protein.

Hybridoma technology was used to produce a set of monoclonal antibodies against a purified polypeptide fragment of type 24 streptococcal M protein to delineate the protective determinants of M protein exposed on the surface of the virulent streptococci. Several hybridoma antibodies were found to be opsonic against the homologous type streptococci. At least two of these antibodies (IIC3.7 and IIC4.6) protected mice against challenge infections with the homologous, but not a heterologous, serotype of bacteria. One of the hybridoma antibodies that reacted in high dilution (1:204,800) with the isolated M protein failed to react with the M protein on the surface of type 24 streptococci, and thus did not opsonize the homologous organisms or protect mice against challenge infections. Because hybridoma antibodies are directed against a single distinct immunodeterminant, these results indicate that protective immunity may be directed at any one of several distinct antigenic determinants of M protein exposed on the surface of virulent group A streptococci.

Animals↗

PubMed1982

Fibronectin-mediated binding of group A streptococci to human polymorphonuclear leukocytes.

Human plasma was shown to promote the attachment of an avirulent strain of Streptococcus pyogenes to human polymorphonuclear leukocytes. Removal of the fibronectin by absorption of the plasma with agarose-immobilized gelatin abolished the attachment-promoting activity. The activity of the absorbed plasma was restored with purified human serum fibronectin. The fibronectin was found to promote the attachment of three M protein-negative, but not of three M protein-positive, strains of streptococci. Radiolabeled fibronectin was shown to bind in various degrees to all strains of S. pyogenes tested. Thus, in the absence of streptococci surface M protein, fibronectin promotes the association of group A streptococci with human polymorphonuclear leukocytes.

Antigens, Bacterial↗