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Biomedical subjects

M R Thompson

Publications and source records attributed to M R Thompson.

At least 37 records · Page 2Linked to original sources

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2 MR/LRP) is a large cell-surface glycoprotein consisting of a 515-kDa and an 85-kDa polypeptide; this receptor is thought to be responsible for the binding and endocytosis of activated alpha 2-macroglobulin and apoE-enriched beta-very low density lipoprotein. A similar high molecular weight glycoprotein has been identified as a potential receptor for Pseudomonas exotoxin A (PE). We demonstrate that the alpha 2 MR/LRP and the PE-binding glycoprotein have a similar mobility upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are immunologically indistinguishable. Furthermore, affinity-purified alpha 2 MR/LRP binds specifically to PE but not to a mutant toxin defective in its ability to bind cells. The 39-kDa receptor-associated protein, which blocks binding of ligands to alpha 2 MR/LRP, also prevents binding and subsequent toxicity of PE for mouse fibroblasts. The concentration of receptor-associated protein that was required to reduce binding and toxicity to 50% was approximately 14 nM, a value virtually identical to the KD measured for the interaction of receptor-associated protein with the purified receptor. Overall, the studies strongly suggest that the alpha 2 MR/LRP is responsible for internalizing PE.

ADP Ribose Transferases↗

Isolation and characterization of Pseudomonas aeruginosa exotoxin A binding glycoprotein from mouse LM cells.

A Pseudomonas aeruginosa exotoxin A (PE) binding glycoprotein was affinity purified from toxin sensitive mouse LM cells. The binding protein was solubilized with Triton X-100 or Nonidet P-40 and purified on a PE-Sepharose affinity column. Polyacrylamide gel electrophoresis yielded a single band with an estimated molecular mass of greater than 300,000 Da. N-Linked carbohydrate was present, accounting for approximately 10% of the total mass of the molecule. The purified protein specifically bound PE. Incubation of purified protein specifically bound PE. Incubation of purified PE binding protein with toxin reduced toxicity to LM cells. We speculate on the role of this toxin binding glycoprotein in the intoxication process.

ADP Ribose Transferases↗

Mouse liver contains a Pseudomonas aeruginosa exotoxin A-binding protein.

The opportunistic pathogen Pseudomonas aeruginosa produces several potential virulence factors, including the ADP-ribosylating toxin, exotoxin A (PE). Studies using a burned mouse model have shown that PE consistently inhibits protein synthesis and depletes elongation factor 2 in mouse liver and variably in other organs. One reason for toxin sensitivity could be the presence of a PE receptor on the surface of cells. Therefore we examined detergent extracts of mouse tissues for the presence of toxin-binding proteins. Proteins which specifically bind PE were present in extracts from liver, kidney, lung, spleen, and heart. Because liver appears to be a prominent target for the toxin in a burned animal, we choose to isolate the PE-binding protein from mouse liver and compare this protein to the recently characterized toxin-binding protein from toxin-sensitive mouse LM fibroblasts. The toxin-binding proteins from both sources have a molecular mass of approximately 350 kDa, share similar protease digestion profiles, and are glycosylated. However the glycosylation patterns for the two species are quite different. Both glycoproteins bind toxin with high avidity. The toxin-binding moiety is located, at least in part, on the plasma membrane and thus could represent the receptor involved in internalization of toxin molecules responsible for cell death.

ADP Ribose Transferases↗

Preparation of stable 125I cyclic GMP tyrosine methyl ester suitable for 3',5' cyclic GMP radioimmunoassay by HPLC.

Determination of the concentration of cyclic guanosine monophosphate (cGMP) by radioimmunoassay (RIA) depends upon the availability of suitable radiolabeled tracers and antibody to detect the product. Reverse phase chromatographic techniques can easily separate the reaction products of chloramine-T iodination of succinyl cGMP tyrosine methyl ester. The binding characteristics of the iodination reaction products to anti-cGMP antibody have been determined. Purified succinyl cyclic nucleotide 125I-tyrosine methyl ester binds to cGMP antisera identically as commercially available tracer. The tracer is stable for greater than three months.

Chromatography, High Pressure Liquid↗

Mutants of staphylococcal toxic shock syndrome toxin 1: mitogenicity and recognition by a neutralizing monoclonal antibody.

Toxic shock syndrome toxin 1 (TSST-1), a 22-kilodalton protein made by strains of Staphylococcus aureus harboring the chromosomal toxin gene, may elicit toxic shock syndrome in humans. In vitro, TSST-1 induces T cells to proliferate and macrophages to secrete interleukin-1. To conduct a structure-function analysis, point mutations on the TSST-1 gene were generated by site-directed mutagenesis to identify amino acids critical for activity of the toxin. Specific tyrosine and histidine residues were replaced by alanines. Wild-type and mutant TSST-1 gene constructs were expressed in Escherichia coli, and the products were tested for their mitogenic potential and reactivity with a TSST-1 neutralizing monoclonal antibody (MAb 8-5-7). Four of the mutants were similar to the wild type; i.e., the mutant toxins stimulated murine T cells and reacted with MAb 8-5-7 equally as well as the wild type. Two mutants exhibited a decrease in mitogenic activity, but one of these retained the capacity to bind with MAb 8-5-7 while the other was no longer recognized by the same antibody. One double mutant demonstrated minimal mitogenic activity and did not react in enzyme-linked immunosorbent and immunoblot assays with MAb 8-5-7. The data show that specific residues near the carboxy terminus of TSST-1 are essential for mitogenic activity and in forming the epitope recognized by neutralizing MAb 8-5-7.

Animals↗

Different crosslinking agents identify distinctly different putative Escherichia coli heat-stable enterotoxin rat intestinal cell receptor proteins.

The receptor for heat-stable enterotoxins (ST) produced by Escherichia coli and related organisms is located in the brush border region of intestinal villus cells. Heterobifunctional and homobifunctional crosslinkers were used to covalently couple 125I-ST to rat intestinal cell brush border membrane proteins. Experimental conditions during ligand binding and subsequent crosslinking significantly influence the efficiency of crosslinking, and the number of peptides specifically crosslinked to the 125I-ST. Multiple proteins efficiently coupled to 125I-ST with agents that can couple through the ST amino terminus. The crosslinker 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC), which can react with the carboxy terminus of the ST, covalently crosslinked 125I-ST to a single protein with an apparent Mr of 125,000-130,000, larger than the proteins identified using longer crosslinkers. Each of the proteins identified by crosslinking migrate with the same retention time on gel filtration after solubilization, with an approximate molecular size of 150,000-200,000.

Azides↗

Citrobacter freundii produces an 18-amino-acid heat-stable enterotoxin identical to the 18-amino-acid Escherichia coli heat-stable enterotoxin (ST Ia).

We purified and sequenced the heat-stable enterotoxin produced by Citrobacter freundii. The toxin was detected during purification by reaction with monoclonal antibody to Escherichia coli heat-stable enterotoxin. The C. freundii toxin amino acid sequence was identical to that of the 18-amino-acid heat-stable enterotoxin (STa) produced by toxigenic E. coli.

Amino Acid Sequence↗

Differences in jejunal and ileal response to E. coli enterotoxin: possible mechanisms.

Escherichia coli, which produce heat-stable enterotoxin (STa), cause intestinal fluid secretion as a mechanism of diarrhea. To determine the factors that modulate the intestinal secretory response, we first compared the time course of the STa-induced secretion in ligated in situ loops of rat jejunum and ileum. We found that the jejunal secretory response was brief (less than or equal to 30 min) while the ileal response to STa was sustained (greater than or equal to 3 h). We then compared the modification of purified STa that occurred in jejunum and ileum and found a close correlation between the continued presence of unmodified, authentic STa and continued fluid secretion in the ligated-loop model. At both sites alteration of STa was demonstrated by high-performance liquid chromatography profile and brush-border membrane binding activity. However, in the jejunum, the modification of STa was qualitatively different and quantitatively much greater. We conclude that the degree to which STa is inactivated or removed from the intestine correlates with the secretory response observed. Inactivation of STa may be a mechanism by which the host limits its secretory response.

Animals↗

Neutralization of toxic shock syndrome toxin-1 by monoclonal antibodies in vitro and in vivo.

Sixteen monoclonal antibodies (MAbs) directed against toxic shock syndrome toxin-1 (TSST-1) were generated by immunization of mice with purified TSST-1 and subsequent fusion of spleen cells with myeloma cells. Antibody-producing clones, identified by an enzyme-linked immunosorbent assay, were maintained as ascites tumors, and MAbs were purified by protein A chromatography. High-titered clones were further characterized and tested for the ability to neutralize several biological activities of TSST-1. The MAbs, which are of several immunoglobulin subtypes, reacted specifically with purified TSST-1 and TSST-1 present in Staphylococcus aureus culture supernatants. Three MAbs neutralized TSST-1-induced mitogenesis in a dose-dependent manner. Three of eight MAbs tested were able to neutralize induction by TSST-1 of interleukin-1 production by human monocytes. One neutralizing MAb, 8-5-7, was tested for the ability to protect rabbits from a constant infusion of TSST-1. Rabbits given the MAb had an attenuated clinical illness and were protected from the hypocalcemia, lipemia, and hepatic and renal insufficiency seen in control rabbits. Six of seven control rabbits died, compared with only one of seven rabbits treated with MAb 8-5-7. These experiments suggest that MAb 8-5-7 is directed against an antigenic determinant critical to the toxicity of TSST-1 and that the MAbs should be useful as probes in structure-function analyses of the TSST-1 molecule.

Animals↗

Protection of rabbits in an infection model of toxic shock syndrome (TSS) by a TSS toxin-1-specific monoclonal antibody.

An anti-TSST-1-specific monoclonal antibody (MAb 8-5-7) was tested for its protective capacity in a rabbit infection model to toxic shock syndrome (TSS). The challenge strain of Staphylococcus aureus (RN4710), which contained a plasmid encoding TSS toxin-1, was introduced into previously implanted chambers. Purified monoclonal antibody (1.25 mg of immunoglobulin G) administered parenterally 1 day before and 1 day after initiation of infection provided complete protection against the TSS-like syndrome and the mortality which occurred in unprotected rabbits.

Animals↗

Binding of E. coli heat-stable enterotoxin to rat intestinal brush borders and to basolateral membranes.

We studied the binding of E. coli heat-stable enterotoxin (STa) to rat brush borders (BB) and to basolateral membranes (BLM) using a biologically active monoiodinated radioligand [( 125I]STa) and highly enriched BB and BLM preparations free of other significant organelle contamination. Binding of [125I]STa to BB was specific; time-, temperature-, and pH-dependent; saturable; and partially reversible. Nonlabeled toxin competitively inhibited the binding of radioligand to BB in a dose-related manner. Scatchard analysis revealed a single class of receptors with an apparent affinity constant of 8.7 +/- 1.5 X 10(8) l/mol. Binding was not affected by amino acids, sugars, and lectins. Proteolytic enzymes significantly decreased binding, although several did so by modifying the radioligand. Trypsin inhibited binding without modifying the radioligand thus supporting the proteinaceous nature of the receptor. Since the enrichment in binding activity in the BB over the homogenate was significantly lower than the enrichment in sucrase activity, we concluded that binding activity is probably associated with other membranous domains, but direct examination revealed no binding activity on basolateral membranes.

Animals↗

In vitro plantlet formation from embryonic explants of eastern white cedar (Thuja occidentalis L.).

A protocol is described for the in vitro production of plantlets from embryonic explants of eastern white cedar (Thuja occidentalis L.). Bud induction was optimal when embryonic explants were cultured for 20-25 days on half-strength Quoirin and Le Poivre mineral salts containing equimolar concentrations (10(-6) M) of N(6)-benzyladenine and 2-isopentyl adenine. Bud development was achieved in phytohormone-free medium in the presence of activated charcoal. Maximal shoot elongation occurred on half-strength Quoirin and Le Poivre salts, whereas shoot multiplication was optimal on half-strength Bornman's MCM salts in the presence of cytokinin. Hardened shoots, dipped in commercial rooting powder containing indole-3-butyric acid, rooted optimally in mist under non-sterile greenhouse conditions. Both rooting and subsequent plantlet growth was best when Redi-Earth((R)) was used as a substrate. Over 250 plantlets per embryo can be produced annually by this technique.

Journal Article↗

Association and dissociation of Escherichia coli heat-stable enterotoxin from rat brush border membrane receptors.

Escherichia coli heat-stable enterotoxin (ST) binds to receptors on rat intestinal cells and brush border membranes (BBM). We devised experiments to examine the reversibility of ST binding. We found that both 125I-labeled ST and native ST were spontaneously dissociable from the BBM receptor. Radiolabeled ST bound to BBM was also dissociated by the addition of avid goat anti-ST antiserum. Furthermore, using a computer program for analysis of ligand binding, we calculated an apparent Ka of 10(8) liters/mol from competitive inhibition and saturation-binding data. This is significantly lower than the value previously reported by others. Our findings, of a lower Ka and a reversible ST-binding process, suggest that a therapeutic strategy of removing bound ST from its receptor or competing with the enterocyte receptor for unbound ST might be successful in terminating ST-induced secretion.

Animals↗

Escherichia coli heat-stable enterotoxins and their receptors.

The family of ST has grown to include closely related toxins produced by a number of organisms. The core sequence of these toxins can bind specifically and reversibly to a receptor found in the microvillus membranes of the intestinal cell brush border. As a result of a specific binding event, the ST can stimulate fluid secretion via receptor-mediated stimulation of guanylate cyclase. The structure of the ST molecule has been partially obtained through a variety of techniques. It is apparent that in the near future, key amino acids and sequence regions of these toxins will be found that will identify the requirements for toxicity. The ST are ideal probes to study the secretory system of intestinal cells since they are not cytotoxic. The receptor for ST has been extensively studied and its purification and characterization will yield important insight into the reversal of toxin-mediated diarrheas, and possibly into the nonpathological secretory process. As yet, the nature of the 54,000-57,000 and 68,000-75,000 dalton binding proteins identified by cross-linking studies are not known. When purified ST-binding peptides and other components of the secretory cascade will become available, they will provide better insight into the actual dynamics of the ST receptor-guanylate cyclase complex secretory pathway.

Animals↗

Blinded, two-laboratory comparative analysis of Escherichia coli heat-stable enterotoxin production by using monoclonal antibody enzyme-linked immunosorbent assay, radioimmunoassay, suckling mouse assay, and gene probes.

Heat-stable enterotoxin (ST)-producing enterotoxigenic Escherichia coli (ETEC) can be identified by a variety of assays, including the suckling mouse assay (SMA), radioimmunoassay (RIA), polyclonal or monoclonal antibody enzyme-linked immunosorbent assay (ELISA), and DNA hybridization with STh and STp gene probes. To compare the sensitivity and reliability of these assays, 100 coded ETEC and non-ETEC isolates were blindly tested in two independent laboratories. SMA, RIA, and monoclonal ELISA were performed in Cincinnati, Ohio, while gene probe analysis was performed in Baltimore, Md. The method of storage of organisms had a profound effect on the stability of plasmids in certain strains. Hybridization experiments to determine the presence or absence of the enterotoxin gene showed that strains stored on Dorset egg medium at room temperature better retained their plasmids than strains stored frozen in skim milk. Forty-four of the 100 organisms obtained from the skim milk stock were found to produce STa in liquid culture by the RIA, SMA, and monoclonal ELISA (100% agreement). However, 50 of 54 of the strains stored on Dorset egg medium which were originally classified as STa+ or ST+ LT+ (positive for both heat-stable and heat-labile [LT] enterotoxins) were found to produce STa and retain the plasmid by each of these assays. Three additional strains were found which harbored the plasmid but did not elaborate STa by any of the assays (3% discrepancy). The monoclonal antibody ELISA appears to be highly reliable for determination of STa production by ETEC and can be easily scored visually even by untrained personnel. Furthermore, when this STa assay is coupled with a polyclonal antibody assay, it is possible to predict the genotype of STh- and STp-producing organisms.

Animals↗