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G D Armstrong

Publications and source records attributed to G D Armstrong.

At least 37 records · Page 2Linked to original sources
PubMed1993

Hydrophobic binding of pertussis toxin is enhanced by oligosaccharide receptors.

Pertussis toxin is one of several virulence factors produced by Bordetella pertussis, the etiologic agent of whooping cough. Pertussis toxin is an oligomeric A-B class toxin composed of an ADP-ribosyltransferase S1 (A) subunit and a B oligomer containing lectin-like binding domains. The carbohydrate binding specificity of the B oligomer is for sialooligosaccharide sequences expressed on target cell receptors and asparagine-linked glycans found in many serum glycoproteins. Pertussis toxin also has the ability to bind to the inert surfaces of culture tubes. In this report we present data showing that pertussis toxin binding to polypropylene microcentrifuge tubes was enhanced in a time- and concentration-dependent manner by the addition of soluble glycoprotein or oligosaccharide receptor analogs. Evidence obtained using the hydrophilic and hydrophobic surfaces of Gel Bond electrophoresis casting film indicated that receptor-enhanced binding was likely due to hydrophobic interactions. Hydrophobic binding of the isolated B oligomer of pertussis toxin was enhanced only in the presence of high concentrations of glycoproteins. Therefore, the S1 (A) subunit of pertussis holotoxin appears to play a role in receptor-enhanced hydrophobic binding. We propose, therefore, that pertussis toxin binding to its receptors may expose or preferentially orient hydrophobic residues that may contribute to the functional association of the toxin with host cell plasma membranes and delivery of the S1 subunit to its intracellular target.

Cell Membrane↗
PubMed1992

Investigation of the lectin-like binding domains in pertussis toxin using synthetic peptide sequences. Identification of a sialic acid binding site in the S2 subunit of the toxin.

Synthetic peptides corresponding to selected sequences in the S2 and S3 subunits of pertussis toxin were prepared and evaluated for their ability to inhibit the binding of biotinylated pertussis toxin and three biotinylated sialic acid specific plant lectins to fetuin and asialofetuin. The screening results indicated that two regions in the S2 subunit corresponding to amino acids 78-98 and 123-154 inhibited pertussis toxin binding to fetuin at submillimolar concentrations, while S3 sequences corresponding to amino acids 87-108 and 134-154 inhibited pertussis toxin-biotin binding to asialofetuin albeit with lower affinity. These results confirm earlier findings, which suggest that the S2 subunit is responsible for binding sialylated glycoconjugates. This was further confirmed by the ability of S2 peptides to inhibit the binding of the lectins from Maackia amurensis and wheat germ to fetuin. Two additional peptides from the S2 subunit of pertussis toxin corresponding to sequences 9-23 and 1-23 were found to contain within their sequences a 6-amino acid fragment which has strong homology with a sequence in wheat germ agglutinin that has been shown to be a component of the sialic acid binding site as determined by x-ray crystallography. One of these sequences from S2 (9-23) was biotinylated and evaluated for its ability to bind to carbohydrate. Through a series of experiments using fetuin, asialofetuin, asialoagalactofetuin, and simple saccharides, the biotinylated peptide was shown to bind with high affinity to sialic acid-containing glycoconjugates indicating that these sequences within the S2 subunit of pertussis toxin also play an important role in binding sialic acid.

Amino Acid Sequence↗
PubMed1991

Synthesis and characterization of a Pertussis toxin-biotin conjugate.

We prepared a Pertussis toxin-biotin conjugate and found its biological properties to be similar to those of native Pertussis toxin with respect to the hemagglutination, Chinese hamster ovary cell, and lymphocyte proliferation assays. Direct binding to Chinese hamster ovary and Jurkat cells was observed using fluorescence microscopy. Pertussis toxin-biotin was also found to possess similar glycoconjugate binding specificities as those of 125I-labeled Pertussis toxin.

Animals↗
PubMed1991

High-level production of murine interleukin-5 (IL-5) utilizing recombinant baculovirus expression. Purification of the rIL-5 and its use in assessing the biologic role of IL-5 glycosylation.

We describe here a recombinant baculovirus expression system useful for high level production of murine recombinant interleukin-5 (rIL-5). In addition, we describe a single-step technique of purification of the rIL-5 from the baculovirus-infected Sf9 cell supernatants, using an anti-IL-5 affinity column. The baculovirus-derived rIL-5 has physical properties and functional activities in various lymphoid cell assays similar to those of natural T cell-derived IL-5 and reacts with anti-IL-5 antibodies. Finally, the rIL-5 is similar to natural T cell-derived IL-5 in manifesting heterogeneous glycosylation; however, glycosylation does not appear to be necessary for biologic function, at least in a lymphoid cell proliferation assay.

Animals↗
PubMed1991

Investigation of Shiga-like toxin binding to chemically synthesized oligosaccharide sequences.

Shiga-like toxin (SLT)-I and SLT-II/IIc bound to Synsorbs containing synthetic alpha Gal(1-4)beta Gal (P1 disaccharide), alpha Gal(1-4)beta GlcNAc (P1 trisaccharide), or alpha Gal(1-4)beta Gal(1-4)beta Glc (Pk trisaccharide) sequences but not to Synsorbs containing alpha Gal(1-3)beta Gal, alpha Gal(1-3)beta Gal(1-4)beta GlcNAc, or the hydrophobic oligosaccharide linkage arm. SLT-I had a preference for Synsorbs containing trisaccharides, whereas SLT-II/IIc binding was less selective. 125I-labeled SLT-I remained bound to Pk trisaccharide Synsorb in the presence of lactose, galactose, or EDTA but was partially released by acetic acid, guanidine HCl, or a 10% solution of SDS. Vero cells coincubated with Pk trisaccharide Synsorb and SLT I extract were protected from this toxin, whereas Pk trisaccharide Synsorb was much less efficient at neutralizing SLT-II/IIc activity in Vero cell coincubation experiments. The SLT-IIc component was not responsible for the inefficient neutralization. Results suggest that synthetic oligosaccharide sequences related to the P blood group antigens coupled to inert matrices could be useful for rapid diagnosis or possibly therapeutic intervention in enterohemorrhagic Escherichia coli infections.

Animals↗
PubMed1990

Comparison of the lectin-like activity of pertussis toxin with two plant lectins that have differential specificities for alpha (2-6) and alpha (2-3)-linked sialic acid.

In this report we have compared the lectin-like properties of Pertussis toxin with two plant lectins which are known to possess different specificities towards terminal Neu5Ac Gal linkages on glycoconjugates. The hemagglutinin from elderberry bark (Sambucus nigra) has a binding specificity for terminal Neu5Ac alpha (2-6) Gal sequences and was found to bind a series of glycoconjugates with a similar specificity as Pertussis toxin. The binding specificity of Pertussis toxin was different from that of the leukoagglutinin from the seeds of Maackia amurensis which preferentially binds terminal Neu5Ac alpha (2-3) Gal sequences. These observations confirm the specificity of Pertussis toxin for Neu5Ac alpha (2-6) Gal glycoconjugate sequences.

Binding, Competitive↗
PubMed1990

Lymphocyte receptors for pertussis toxin.

We have investigated human T-lymphocyte receptors for pertussis toxin by affinity isolation and photoaffinity labeling procedures. T lymphocytes were obtained from peripheral human blood, surface iodinated, and solubilized in Triton X-100. The iodinated mixture was then passed through pertussis toxin-agarose, and the fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of the fixed, dried gels revealed several bands in the pertussis toxin-bound fraction that were not observed in fractions obtained from histone or fetuin-agarose. Further investigations employed a photoaffinity labeling reagent, sulfosuccinimidyl 2-(p-azido-salicylamido)-1,3'-dithiopropionate, to identify pertussis toxin receptors in freshly isolated peripheral blood monocytic cells, T lymphocytes, and Jurkat cells. In all three cell systems, the pertussis toxin affinity probe specifically labeled a single protein species with an apparent molecular weight of 70,000 that was not observed when the procedure was performed in the presence of excess unmodified pertussis toxin. A protein comparable in molecular weight to the one detected by the photoaffinity labeling technique was also observed among the species that bound to pertussis toxin-agarose. The results suggest that pertussis toxin may bind to a 70,000-Da receptor in human T lymphocytes.

Affinity Labels↗
PubMed1989

Lectinlike properties of pertussis toxin.

We have examined the lectinlike properties of pertussis toxin by binding-inhibition assays and affinity chromatography of goose erythrocyte membranes. Although pertussis toxin and wheat germ agglutinin apparently recognize similar sugar sequences on glycoproteins, the binding activities of the two lectins are not identical.

Acetylglucosamine↗
PubMed1989

Examination of enterotoxigenic Escherichia coli H10407 (colonization factor antigen I+) by scanning electron microscopy with conductive staining.

We have used the scanning electron microscope to examine enterotoxigenic Escherichia coli H10407, which expresses colonization factor antigen I pili. The use of low accelerating voltages and conductive staining procedures allowed us to obtain images of colonization factor antigen I pili and other structural details which were obscured by conventional gold-coating techniques.

Agglutination↗
PubMed1988

Use of glycosyltransferases to restore pertussis toxin receptor activity to asialoagalactofetuin.

Fetuin derivatives with enzymatically altered oligosaccharide units were tested for their ability to inhibit pertussis toxin-mediated agglutination of goose erythrocytes and the binding of 125I-labeled fetuin to pertussis toxin-coated polystyrene tubes. Fetuin oligosaccharides were sequentially degraded by treatment with: neuraminidase (asialofetuin) followed by beta-galactosidase (asialoagalactofetuin) and, lastly, with beta-N-acetylhexosaminidase (asialoagalacto-a[N-acetylglucosamino]fetuin). Asialofetuin retained only 19 and 53% of the inhibitory activity of native fetuin in the hemagglutination and 125I-fetuin binding assays, respectively. Asialoagalactofetuin showed no further reduction of inhibition in the hemagglutination system and, instead, resulted in partial recovery of inhibition in the 125I-fetuin-pertussis toxin binding assay. Asialoagalacto-a[N-acetylhexosamino]fetuin showed a further decrease in ability to inhibit pertussis toxin binding in both assays. The inhibitory activity of asialoagalactofetuin could be restored to that of native fetuin by adding back D-galactose with UDP-Gal:D-glucosyl-1,4-beta-galactosyltransferase, followed by the addition of terminal sialic acid residues with CMP-N-acetylneuraminic acid:beta-D-galactosyl-1,4-N-acetyl-beta-D-glucosamine-alpha-2,6-N- acetylneuraminyltransferase. The data suggested that a requirement for pertussis toxin binding to fetuin may be the presence of acetamido-containing sugar groups in the nonreducing terminal position of fetuin's oligosaccharides.

Animals↗
PubMed1988

The use of lectin affinity columns for selection of precursor or fully glycosylated forms of glycoprotein gD1 of herpes simplex virus type 1.

Several lectins were examined for their ability to bind to the glycoprotein gD1 polypeptide from Vero cells infected by herpes simplex virus type 1 (HVS-1), strain KOS. At least four distinct forms of gD1 (1, 2, 3 and 4), ranging in size from 59K to 52K, were resolved by SDS-10% polyacrylamide gel electrophoresis. Wheat germ agglutinin (WGA) did not bind to any of these forms, suggesting that if any sialic residues are present in the carbohydrate moieties of gD1, they are not available for binding to WGA. The entire population of forms 1 and 2 (approximately 59K) bound to castor bean-120 (CB-120) lectin affinity columns, suggesting the presence of terminal galactose residues on the mature and more fully glycosylated carbohydrate moieties of gD1. The forms 3 and 4, representing precursor gD1 molecules, did not bind. The majority of forms 2 and 4, and a portion of form 3 bound to lentil lectin, suggesting the presence of fucose and alpha-linked mannosyl residues on these molecules. A gD1-specific, high molecular weight species (120-125K) was detected in the lentil lectin-binding fraction but not in the fraction bound to CB-120 lectin or in the original infected-cell extract. The results indicated that lectin affinity chromatography, using lentil and CB-120 lectins, is useful as an initial step for the selection and purification of the individual glycosylated forms of gD1.

Animals↗
PubMed1988

Identification of a human erythrocyte receptor for colonization factor antigen I pili expressed by H10407 enterotoxigenic Escherichia coli.

We have identified a receptor for colonization factor antigen I (CFA/I) pili in human erythrocyte membranes. Erythrocyte binding assays, using whole organisms, suggested that the CFA/I receptor was a glycoprotein containing important sialic acid moieties. Subsequently, human erythrocyte membranes were extracted with lithium diiodosalicylate to obtain a soluble glycoprotein fraction from which to isolate receptors. The extracted material caused agglutination of the CFA/I+ but not the CFA/I- organisms at a protein concentration of 0.5 mg/ml. The CFA/I receptor was identified in iodinated extract by an affinity isolation procedure, using whole bacterial cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the washed, extract-coated H10407 CFA/I+ organisms revealed a band with an apparent molecular weight of 26,000 which was present in the original extract but was not observed on extract-coated H10407 CFA/I- bacteria. The addition of purified CFA/I pili reduced binding of the 26,000-molecular-weight receptor to CFA/I+ bacteria. The CFA/I-specific receptor species also bound to wheat germ agglutinin-agarose. This observation supported the suggestion that the CFA/I receptor identified in this report is a sialoglycoprotein.

Antigens, Bacterial↗
PubMed1987

Maintenance of biological activity of pertussis toxin radioiodinated while bound to fetuin-agarose.

We developed a method to produce radioiodinated pertussis toxin (PT) which was active in the goose erythrocyte agglutination and CHO cell assay systems. The procedure used fetuin coupled to agarose to prevent inactivation of the toxin during the iodination reaction. Analysis of the labeled PT by affinity chromatography on fetuin-agarose and wheat germ agglutinin-agarose and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that there were minimal amounts of labeled fetuin or other contaminants in the labeled PT preparations. All five of the subunits of the toxin appeared to be labeled by the procedure. The labeling method will facilitate further investigations into the nature of the interaction and activity of PT in host tissues.

Animals↗
PubMed1986

Preferred sites on cytochrome c for electron transfer with two positively charged blue copper proteins, Anabaena variabilis plastocyanin and stellacyanin.

Rate constants for the reactions of horse cytochrome c (E'0 of +260 mV) with the copper proteins Anabaena variabilis plastocyanin (E'0 of +360 mV) used as oxidant and stellacyanin (E'0 of +187 mV) used as reductant have been determined at 25 degrees C, pH 7.5 and 7.0, respectively, and an ionic strength of 0.10 M (NaCl). These rate constants were also measured with eight different singly substituted 4-carboxy-2,6-dinitrophenyl (CDNP) horse cytochrome c derivatives, modified at lysine-7, -13, -25, -27, -60, -72, -86, or -87 and with the trinitrophenyl (TNP) derivative modified at lysine-13. The influence of the modifications on the bimolecular rate constants for these reactions defines the region on the protein that is involved in the electron-exchange reactions and demonstrates that the preferred site is at or near the solvent-accessible edge of the heme prosthetic group on the "front" surface of the molecule. Both reactions are strongly influenced by the lysine-72 modification to the left of the exposed heme edge and, to this extent, behave similar to the earlier studied reaction with azurin. These effects span only an order of magnitude in rate constants and are thus many times smaller than those for the physiological protein redox partners of cytochrome c. While the preferred sites of reaction on the surface of cytochrome c for small inorganic complexes appear to be dependent only on the net charge of the reactants, with the copper proteins additional factors intervene. These influences are discussed in terms of hydrophobic patches and the distribution of charges on the surface of the four copper proteins so far examined.

Binding Sites↗
PubMed1986

Home care for children dying of cancer.

The feasibility of home care as an alternative to hospitalization for children dying of cancer was studied. The home care system was defined as nurse-directed with a consultant physician and did not entail extensive participation by other health professionals. Of 58 children cared for at home during the 2-year project, 79% died at home and 21% died in the hospital or en route to it. The findings, as shown by interview data, suggest that home care at the end stage of life is a viable alternative for children dying of cancer and for their families.

Adolescent↗
PubMed1986

Location of the antigenic determinants of conjugative F-like pili.

The amino terminus of the pilin protein constitutes the major epitope of F-like conjugative pili studied to date (F, ColB2, R1-19, R100-1, and pED208). Anti-pED208 pilus antibodies were passed through a CNBr-Sepharose affinity column linked to bovine serum albumin which was conjugated to a synthetic peptide, AcP(1-12), containing the major epitope at the amino terminus of pED208 pilin. This allowed the separation of two classes of antibodies; one was specific for the amino terminus and bound to the column, while the other, which recognizes a second epitope on the pilus, did not bind to the column. In addition, antibodies were raised against two amino-terminal peptide-bovine serum albumin conjugates [AcP(1-8) and AcP(1-12)] to ensure a source of pure, high-titer antibodies directed against the amino terminus. The location of these antibodies on intact pili was assayed by immunoelectron microscopy with a protein A-gold technique. The amino terminus-specific antibodies did not bind to the sides of the pili but appeared to be associated with the pilus tip. In addition, these antibodies were found to bind to the vesicle-like structure at the base of the pilus. The anti-pilus antibodies not specific for the amino terminus (unbound immunoglobulin G) were found to bind to the sides of the pilus. Anti-F and anti-ColB2 pilus antibodies bound to the sides of F, ColB2, and R1-19 pili, which have only their secondary epitope in common. The carboxyl-terminal lysine of R1-19 pilin prevents the absorption of anti-F plus antiserum but not anti-ColB2 pilus antiserum to the sides of the pilus, presumably by interfering with the recognition of this secondary epitope.

Antibodies, Bacterial↗
PubMed1984

Studies on reovirus receptors of L cells: virus binding characteristics and comparison with reovirus receptors of erythrocytes.

"Saturation binding experiments" were carried out to characterize the attachment of reovirus to mouse L fibroblasts. Scatchard analysis of data obtained from such experiments suggests that one homogeneous set of noncooperative, high affinity binding sites are involved in reovirus attachment. It is estimated that L cells possess between 3 and 5 X 10(5) reovirus binding sites per cell and that the equilibrium dissociation constant (KD) is approximately 3 X 10(-9) M. Scatchard analysis of data from similar experiments, carried out in the presence of anti-sigma 1 and anti-sigma 3 antibodies, revealed that although both antibodies prevent viral attachment, they exhibit distinct binding inhibition characteristics: anti-sigma 1 effectively abolishes high-affinity, specific binding, whereas anti-sigma 3 apparently blocks low-affinity, nonspecific interactions. The nature of the L-cell receptor was then probed using various enzymes and reagents, and compared with that of the reovirus receptor on human type O erythrocytes. It was found that whereas reovirus hemagglutination (HA) is inhibited by pretreatment of erythrocytes with various proteases or neuraminidase, virus binding to L cells is unaffected by such treatments. Neither HA nor cell binding is inhibited by the various sugars tested, including N-acetyl-D-glucosamine, which was previously reported to inhibit reovirus HA (L. D. Gelb and A. M. Lerner, 1965, Science 147, 404-405). Both L cells and erythrocyte reovirus receptors are nevertheless highly sensitive to periodate treatment, which presumably destroys the high-affinity reovirus binding sites since protein sigma 1, which is capable of attaching to L cells by itself, does not bind to cells pretreated with periodate. It is therefore concluded that sugar residues on the receptor may be involved in this specific interaction. The possibility that gangliosides may serve as reovirus receptors was also probed. It was found that bovine brain gangliosides, but not cerebrosides, readily aggregate reovirus, inhibit HA, and block viral attachment to L cells. However, binding of protein sigma 1 to L cells is unaffected by gangliosides. Inhibition of reovirus HA and L-cell binding by these gangliosides is therefore most likely due to a steric hindrance effect brought about by interactions between the gangliosides and other components of the outer viral capsid.

Animals↗