Biomedical subjects

G S Chhatwal

Publications and source records attributed to G S Chhatwal.

At least 73 records · Page 4Linked to original sources

PubMed1990

Role of complement S protein (vitronectin) in adherence of Streptococcus dysgalactiae to bovine epithelial cells.

The binding of bovine complement S protein (vitronectin) to Streptococcus dysgalactiae isolates from cattle with mastitis and the S protein's role in streptococcal adherence to bovine epithelial cells were investigated. All 25 clinical isolates of S dysgalactiae interacted with bovine S protein. None of the other streptococcal species tested bound to bovine S protein. The S protein-binding sites were saturable and highly sensitive to trypsin. The binding of bovine S protein to S dysgalactiae isolates was specific and could not be inhibited by other plasma proteins, such as fibronectin, albumin, fibrinogen, alpha 2-macroglobulin, or IgG. Similarly, streptococcal binding of bovine S protein was not influenced by the synthetic peptide Gly-Arg-Gly-Asp-Ser, which constituted the host cell attachment sequence of S protein. In adherence experiments, prior binding of bovine S protein to S dysgalactiae enhanced streptococcal adherence to bovine epithelial cells. The enhancing effects by bovine S protein were abolished when the respective binding sites on the streptococci were digested by trypsin. Thus, bovine S protein could be an important mediator of adherence of S dysgalactiae to bovine epithelial cells.

Animals↗

PubMed1988

Botulinum ADP-ribosyltransferase C3. Purification of the enzyme and characterization of the ADP-ribosylation reaction in platelet membranes.

A novel ADP-ribosyltransferase C3 was purified to homogeneity from filtrates of certain strains of Clostridium botulinum type C by ammonium sulfate precipitation, gel filtration, ion-exchange chromatography and heat treatment. The molecular mass of botulinum ADP-ribosyltransferase C3 was found to be 25 kDa. In the presence of [32P]NAD but not with [carbonyl-14C]NAD, C3 labelled 21-24-kDa protein(s) in membranes of human platelets and other tissues. The Km value of the ADP-ribosylation reaction for NAD was about 2 microM. Labelling of the 21-24-kDa protein(s) by C3 was largely reduced by addition of nicotinamide. Snake venom phosphodiesterase cleaved the ADP-ribose attached to the 21-24-kDa protein(s) by C3 and released 5'AMP. C3 catalyzed hydrolysis of [carbonyl-14C]NAD and released [carbonyl-14C]nicotinamide. ADP-ribosylation of 21-24-kDa platelet membrane protein(s) was biphasically regulated by Mg2+, Mn2+ and Ca2+. In the absence of free divalent cations GTP, GTP[gamma S] and GDP but not GDP[beta S], GMP, ATP or ATP[gamma S] increased labelling by C3. In the presence of Mg2+, GTP[gamma S] was inhibitory. Guanine nucleotides prevented heat inactivation of the substrate protein(s) with the rank order GTP[gamma S] = GTP = GDP greater than GDP[beta S] greater than GMP much greater than ATP = GMP = ATP[gamma S]. The data support the view that the novel ADP-ribosyltransferase C3 modifies eukaryotic 21-24-kDa GTP-binding protein(s).

ADP Ribose Transferases↗

PubMed1988

ADP-ribosylation of skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin.

The enzymatically active component ia of Clostridium perfringens iota toxin ADP-ribosylated actin in human platelet cytosol and purified platelet beta/gamma-actin, in a similar way to that been reported for component I of botulinum C2 toxin. ADP-ribosylation of cytosolic and purified actin by either toxin was inhibited by 0.1 mM phalloidin indicating that monomeric G-actin but not polymerized F-actin was the toxin substrate. Perfringens iota toxin and botulinum C2 toxin were not additive in ADP-ribosylation of platelet actin. Treatment of intact chicken embryo cells with botulinum C2 toxin decreased subsequent ADP-ribosylation of actin in cell lysates by perfringens iota or botulinum C2 toxin. In contrast to botulinum C2 toxin, perfringens iota toxin ADP-ribosylated skeletal muscle alpha-actin with a potency and efficiency similar to non-muscle actin. ADP-ribosylation of purified skeletal muscle and non-muscle actin by perfringens iota toxin led to a dose-dependent impairment of the ability of actin to polymerize.

ADP Ribose Transferases↗

PubMed1988

Mediation of adherence of streptococci to human endothelial cells by complement S protein (vitronectin).

The role of S protein in the adherence of group A and G streptococci to human umbilical vein endothelial cells cultivated in 96-well microdilution plates was studied by utilizing fluorescein-labeled streptococci. The assay proved suitable for quantitative determination of bacterial adherence to cultured endothelial cells for all tested strains of streptococci. Only bacterial strains with significant S protein binding but weak fibronectin binding were included in these studies. Fibronectin-mediated adherence to endothelial cells of these streptococci was less than 25% of total and could be blocked by antifibronectin immunoglobulin G. Further treatment of endothelial cell monolayers with anti-S protein immunoglobulin G at concentrations up to 1 mg per well led to an almost complete inhibition of adherence for all tested streptococcal cultures, indicating significant contribution of S protein in the streptococcus-endothelial cell interaction. Blocking of S-protein-binding sites on streptococci by preincubation with exogeneous S protein at a concentration of 10 micrograms per 4 x 10(7) streptococci led to about 75% reduction of S-protein-mediated adherence to endothelial cells. Trypsin pretreatment of group G streptococci and pronase pretreatment of group A and G streptococci, modifications known to destroy the bacterial binding sites of S protein, also inhibited the capacity of the streptococci for S-protein-mediated adherence to endothelial cells by 75 to 80%. These results indicate that S protein plays a mediatory role in adherence of streptococci to endothelial cells and that S-protein-specific binding sites on streptococci are involved in this interaction.

Bacterial Adhesion↗

PubMed1988

Adherence of streptococcal isolates from cattle and horses to their respective host epithelial cells.

Adherence of Streptococcus dysgalactiae isolates from cattle and S equi isolates from horses to their respective host epithelial cells was compared with the adherence of S pyogenes to human epithelial cells. The adherence was quantitatively determined by use of fluorescein-labeled streptococci. All 3 streptococcal species adhered selectively to their respective host cells. The mechanism of adherence was evaluated by binding studies with adhesive plasma protein, fibronectin. Although all 3 streptococcal species bound fibronectin, S dysgalactiae and S equi interacted preferentially with a 210-kilodalton (kD) C-terminal fragment of fibronectin, whereas S pyogenes bound only a 29-kD N-terminal fragment. A synthetic peptide Gly-Arg-Gly-Asp-Ser, representing the host cell attachment site of fibronectin, partially inhibited the binding of fibronectin and of its 210 kD fragment to S dysgalactiae, but not to S equi. The binding of fibronectin and its 29-kD fragment to S pyogenes was not inhibited by Gly-Arg-Gly-Asp-Ser. These differences in binding activities corresponded to the ability of fibronectin to mediate the adherence of the streptococci to the epithelial cells: fibronectin strongly inhibited the adherence of S pyogenes and S equi to the epithelial cells, but only weakly inhibited that of S dysgalactiae.

Animals↗

PubMed1987

Botulinum ADP-ribosyltransferase C3 but not botulinum neurotoxins C1 and D ADP-ribosylates low molecular mass GTP-binding proteins.

Botulinum ADP-ribosyltransferase C3 modified 21-24 kDa proteins in a guanine nucleotide-dependent manner similar to that described for botulinum neurotoxin C1 and D. Whereas GTP and GTP gamma S stimulated C3-catalyzed ADP-ribosylation in the absence of Mg2+, in the presence of added Mg2+ ADP-ribosylation was impaired by GTP gamma S. C3 was about 1000-fold more potent than botulinum C1 neurotoxin in ADP-ribosylation of the 21-24 kDa protein(s) in human platelet membranes. Antibodies raised against C3 blocked ADP-ribosylation of the 21-24 kDa protein by C3 and neurotoxin C1 but neither cross reacted with neurotoxin C1 immunoblots nor neutralized the toxicity of neurotoxin C1 in mice. The data indicate that the ADP-ribosylation of low molecular mass GTP-binding proteins in various eukaryotic cells is not caused by botulinum neurotoxins but is due to the action of botulinum ADP-ribosyltransferase C3. The weak enzymatic activities described for botulinum neurotoxins appear to be due to the contamination of C1 and D preparations with ADP-ribosyltransferase C3.

ADP Ribose Transferases↗

PubMed1987

Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin.

The culture medium of certain strains of Clostridium botulinum type C contains two separable ADP-ribosyltransferases. Besides the ADP-ribosylation of actin due to botulinum C2 I toxin, a second microbial enzyme causes the mono-ADP-ribosylation of a eukaryotic protein with a molecular mass of about 20 kDa found in platelets, neuroblastoma X glioma hybrid cells, S49 lymphoma cells, chick embryo fibroblasts and sperm. The eukaryotic substrate is inactivated by heating and trypsin treatment. In contrast, the novel ADP-ribosyltransferase, which can be separated by DEAE-Sephadex chromatography, is largely resistant in the short term to trypsin digestion.

ADP Ribose Transferases↗

PubMed1987

Isolation and properties of a novel IgG-binding protein from streptococci of serological group U.

A nonimmune binding of immunoglobulin (Ig) G has been detected in streptococci of group U. The group U Fc-binding site differed from the five previously known types of staphylococcal and streptococcal Fc-binding sites by its strong affinity for murine IgG, with dissociation constants in nanomolar range for rat and mouse IgG, as well as for mouse IgG subclasses 1, 2a, 2b and 3. It also differed from other binding sites by the high sensitivity towards trypsin. The Fc-binding protein could be solubilized from the streptococci of group U with papain and purified by gel filtration on sephacryl S-200 and by subsequent affinity chromatography on human IgG-Sepharose. The purified binding protein was homogeneous on SDS-polyacrylamide gel electrophoresis and had a molecular weight of approximately 58,000 daltons. It retained its binding activities for murine IgG subclasses as revealed by western blotting. Coupled to CNBr-activated sepharose, the purified Fc-binding protein could be effectively used for the isolation of murine IgG subclasses by affinity chromatography.

Animals↗

PubMed1987

Heterogeneity of fibronectin reactivity among streptococci as revealed by binding of fibronectin fragments.

Streptococci belonging to serological groups A, B, C, G, L and U were studied for their interaction with 125I-labelled fibronectin and its fragments. Fibronectin purified from humans plasma by affinity chromatography on gelatin-agarose and heparin-agarose was cleaved by thrombin into a 29,000 Dalton and a 210,000 Dalton fragments. Terminal analysis of purified fragments indicated that 29,000 fragment was from amino-terminal and 210,000 fragment from carboxyl-terminal domain of fibronectin. Binding of fibronectin was observed in all streptococci except those of group B. Streptococci of groups A, G, L, U and S. equisimilis reacted only with 29,000 Dalton fragment whereas S. dysgalactiae, S. zooepidermicus and S. equi reacted only with 210,000 Dalton fragment. The streptococcal binding sites for these two fragments were distinct from each other. Fibrinogen blocked the binding of 210,000 Dalton fragment but not of 29,000 Dalton fragment. Trypsinization of streptococci did not affect their binding sites for 210,000 Dalton fragment but destroyed those for 29,000 Dalton fragment. The results indicate that the streptococci of group A and G as well as S. equisimilis which are mainly pathogenic in humans bind amino-terminal fragment of fibronectin. This may facilitate the adherence of these pathogens. On the other hand, the streptococci isolated from animal infections had different binding sites recognized only by carboxyl-terminal part of fibronectin.

Binding Sites↗

PubMed1987

A simple method for quantitative determination of bacterial adherence to human and animal epithelial cells.

A simple quantitative method for determining bacterial adherence to epithelial cells was devised. The method involved incubation of fluorescein-labeled bacteria with oral epithelial cells. Non-adherent bacteria were subsequently removed by Percoll density centrifugation. The degree of bacterial adherence was then determined fluorospectrophotometrically by measuring the fluorescein extracted from the adherent bacteria. The degree of adherence corresponded well with the microscopic counts of adherent bacteria. The method proved to be suitable for assaying adherence of both gram-positive and gram-negative bacteria to human as well as animal epithelial cells.

Animals↗

PubMed1987

Specific binding of the human S protein (vitronectin) to streptococci, Staphylococcus aureus, and Escherichia coli.

Specific binding of the 125I-labeled human S protein (vitronectin) which has been shown to be identical with serum-spreading factor, was observed with group A, C, and G streptococci as well as with Staphylococcus aureus and Escherichia coli. The specific binding of S protein to group A, C, and G streptococci was high, whereas the binding to S. aureus and E. coli cultures was moderate. In contrast, group B streptococci and a number of other bacterial species tested did not interact with S protein. The binding of S protein to bacteria was saturable and could be inhibited only by unlabeled S protein but not by albumin. Trypsinization and heat treatment of bacteria destroyed the S-protein binding capacity for group G streptococci, S. aureus, and E. coli but not for group A and C streptococci. Likewise, unlabeled human fibronectin and heparin inhibited the binding of labeled S protein to group G streptococci, S. aureus, and E. coli, but did not influence the binding to group A and C streptococci. Double-reciprocal plots of S-protein binding to group G streptococci indicated that fibronectin inhibited the binding in a competitive manner, while heparin acts in a noncompetitive manner. Moreover, the binding of S protein to G streptococci could be partially by the synthetic peptide Gly-Arg-Gly-Asp-Ser, which contains the cell attachment site of S protein. Trypsin-treated S protein had similar binding activity as untreated S protein for group G streptococci, S. aureus, and E. coli, but showed reduced binding to group A and C streptococci. The present data are indicative of two different types of bacterial binding sites in S protein. The binding to group G streptococci, S. aureus, and E. coli is mediated in part through a domain in the S protein containing the sequence Arg-Gly-Asp, whereas a different site is responsible for the binding to group A and C streptococci.

Bacterial Adhesion↗

PubMed1987

Novel complex formed between a nonproteolytic cell wall protein of group A streptococci and alpha 2-macroglobulin.

Binding of 125I-labeled alpha 2-macroglobulin (alpha 2M) to streptococci belonging to serological groups A, B, C, and G was studied. Streptococci of groups A and G interacted only with native alpha 2M, and those of group C reacted only with alpha 2M-trypsin complex. Binding of alpha 2M to group A streptococci was saturable and reversible. The dissociation constant was 2.02 X 10(-7) M, and the number of binding sites was calculated to be 18,000 per streptococcus. The alpha 2M-binding protein could be solubilized by treatment of group A streptococci with a murolytic enzyme and subsequently purified by affinity chromatography and high-pressure liquid chromatography. The purified protein was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular weight of 78,000. It possessed no proteolytic activity and interacted with native alpha 2M in Western blots (immunoblots). Interaction of purified binding protein with alpha 2M led to a change in the conformation of alpha 2M similar to that obtained by alpha 2M-protease complexes. Reversible binding of a nonproteolytic streptococcal component of alpha 2M is thus a novel feature of alpha 2M reactivity.

Bacterial Proteins↗

PubMed1986

Release of Fc-receptors after streptococcal lysis induced by a lytic enzyme from Streptomyces globisporus.

Streptomyces globisporus produced an enzyme which had lytic effects on streptococci and was able to solubilize their Fc-receptors. A new method for rapid purification of this enzyme was described. It consisted of adsorption to Amberlite CG 50 and subsequent chromatography on CM-cellulose Whatman CM 52. The purified enzyme was free of proteases and had lysed streptococci of serological groups A, B, C, G and L. It released Fc-receptors from the streptococcal surface in a biologically active form.

Enzymes↗

PubMed1986

Specific binding sites for monomeric and aggregated beta 2-microglobulin on surface of groups A, B, C, and G streptococci.

Human beta 2-microglobulin (beta 2-m) was isolated from urine samples of patients with tubular dysfunctions and aggregated with glutaraldehyde. Four aggregates with molecular weights of 800,000, 480,000, 260,000, and 60,000 were separated by filtration on Sephacryl S-300. The aggregates and monomeric beta 2-m (11,800 MW) were subsequently labeled with 125I and tested for binding to streptococci. Group A streptococci bound only aggregated beta 2-m with a mean binding of 44.5%. Most of the group G streptococci, on the other hand, bound only monomeric beta 2-m with a mean binding of 58%. Among group B streptococci the serotypes with protein antigens interacted mainly with monomeric beta 2-m and those without protein antigens preferentially with aggregated beta 2-m. Nontypable group B streptococcal serotypes did not bind monomeric or aggregated beta 2-m. Of the streptococci belonging to group C, S. equisimilis reacted with monomeric beta 2-m and S. dysgalactiae with aggregated beta 2-m. S. equi did not interact with monomeric beta 2-m or aggregated beta 2-m. Bindings of monomeric beta 2-m and aggregated beta 2-m were saturable and could be inhibited by the respective unlabeled forms of beta 2-m. Fibrinogen, fibronectin, alpha 2-macroglobulin, haptoglobin, or immunoglobulin G did not inhibit the binding of either form of beta 2-m. The binding sites for monomeric beta 2-m were more susceptible to trypsin than those for aggregated beta 2-m. Treatment of streptococci with pronase destroyed their binding activities for monomeric and aggregated beta 2-m. Both monomeric beta 2-m and aggregated beta 2-m binding sites were sensitive to heat. The Scatchard plots of monomeric beta 2-m and aggregated beta 2-m were linear with Kd of 1.29 X 10(-9) M and 1.9 X 10(-9) M respectively. The number of binding sites per bacterium were estimated to be 81,000 for monomeric beta 2-m and 1,210 for aggregated beta 2-m.

Binding Sites↗

PubMed1985

Interactions of plasma proteins with group A, B, C and G streptococci.

Streptococci of serological groups A, B, C and G displayed different binding activities for plasma proteins. Most of the streptococci studied, except those of group B, bound immunoglobulin G. All streptococci reacted with fibrinogen and, except those of group B, with fibronectin. The majority of streptococci, but none of group B, had an affinity for alpha 2-macroglobulin. Albumin was bound by all cultures of group G and a few of group C. Haptoglobulin interacted with only 1 group A culture. None of the streptococci bound transferrin. The specificity of binding sites for 125I-labelled plasma proteins was revealed in a series of inhibition experiments with the unlabelled proteins. The binding sites on streptococci of group G showed different sensitivities to trypsin and pepsin. Reactivities for immunoglobulin G, however, remained unaffected after treatments of the streptococci with trypsin. Exposure to heat (30 min, 80 degrees C) partially inactivated binding activities for the plasma proteins. Sodium dodecyl-sulphate and acetylimidazole strongly reduced binding of albumin and to a lesser extent that of alpha 2-macroglobulin. They had no or little effect on the interaction with the other plasma proteins. Dioxane decreased almost all binding activities. Ethanol partially diminished the binding of immunoglobulin G, fibrinogen, fibronectin and alpha 2-macroglobulin. Treatments of group G streptococci with guanidine, urea, formamide or methanol-HC1 did not affect their plasma protein binding activities.

Animals↗

PubMed1985

Interactions of immunoglobulin G, fibrinogen and fibronectin with Staphylococcus hyicus and Staphylococcus intermedius.

Binding of immunoglobulin G, fibrinogen and fibronectin to 112 cultures of coagulase-positive staphylococci together with 7 of coagulase-negative S. hyicus subsp. chromogenes were investigated. Of the coagulase-positive staphylococcal cultures 45 were S. hyicus subsp. hyicus, 51 S. intermedius and 16 S. aureus. All 45 S. hyicus subsp. hyicus cultures coagulated plasma preparations from pigs and not always those from sheep, rabbits and dogs. Labelled IgG was bound by all cultures of S. hyicus subsp. hyicus and S. aureus, but only by 6 of 51 S. intermedius cultures. Fibrinogen interacted with 28 of the 45 S. hyicus subsp. hyicus cultures, with 17 of the 51 S. intermedius cultures and with S. aureus throughout. Fibronectin reacted with 19 cultures of S. hyicus subsp. hyicus, 11 of S. intermedius and all S. aureus. The binding activities for labelled IgG were more pronounced than those for fibrinogen and fibronectin. None of the 7 cultures of S. hyicus subsp. chromogenes bound any of these plasma proteins. Bindings of fibrinogen and fibronectin to S. hyicus subsp. hyicus and S. intermedius elicited only in part distinct clumping reactions of the staphylococci in the respective plasma proteins.

Animals↗

PubMed1985

Fibrinogen binding inhibits the fixation of the third component of human complement on surface of groups A, B, C, and G streptococci.

Effects of fibrinogen binding to M protein-positive and -negative streptococci on fixation of the third component of human complement (C3) were determined. In all test cultures of serological groups A, B, C, and G fixation of C3 was observed in normal human serum as revealed by quantitative fluorescent immunoassay. Fibrinogen binding inhibited the fixation of C3 on streptococci. The degree of inhibition was proportional to the extent of fibrinogen binding. Thus, inhibition of C3 fixation was most pronounced in strongly fibrinogen-positive streptococci of groups A, C, and G and not demonstrable in fibrinogen-negative cultures of groups C and G. Trypsinization of the streptococci destroyed their capacity to bind fibrinogen and consequently the inhibitory effects on C3 fixation. The carboxymethylated alpha and beta chains of fibrinogen moderately inhibited C3 fixation whereas gamma chain had no influence. These studies may indicate that fibrinogen binding structures other than M protein could also be involved in streptococcal pathogenicity.

Binding Sites↗

PubMed1985

Binding of alpha 2-macroglobulin and haptoglobin to Actinomyces pyogenes.

All 25 cultures of Actinomyces pyogenes tested in the present study bound 125I-labelled human alpha 2-macroglobulin with a mean binding of 65.6%. Thirteen cultures also bound 125I-labelled human haptoglobin with a mean of 51.5%. None interacted with fibrinogen, fibronectin, immunoglobulin G, or albumin. Twenty-eight cultures representing other species of actinomycetaceae did not show any interaction with alpha 2-macroglobulin, haptoglobin, and other plasma proteins tested. The binding of alpha 2-macroglobulin and haptoglobin to A. pyogenes was saturable and could be completely inhibited by the respective unlabelled plasma proteins. The binding of alpha 2-macroglobulin could not be inhibited by unlabelled haptoglobin. On the other hand, alpha 2-macroglobulin blocked the binding of haptoglobin, possibly by steric hindrance. Treatment of the bacteria with trypsin reduced their binding activities for alpha 2-macroglobulin and haptoglobin indicating the protein nature of the binding sites. Exposure to heat (1 h, 80 degrees C) significantly diminished the binding activity for haptoglobin, but not that for alpha 2-macroglobulin. The binding of alpha 2-macroglobulin and haptoglobin could be an important feature in the classification of A. pyogenes among the members of actinomycetaceae.

Actinomyces↗