Generation of cell surface neoganglioproteins. GM1-neoganglioproteins are non-functional receptors for cholera toxin

GM1 (II3Neu5Ac-GgOse4Cer)-oligosaccharide was prepared from the ganglioside by ozonolysis and alkaline fragmentation, reductively aminated and coupled to the heterobifunctional cross-linker succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate. The resulting derivative reacted with free sulfhydryl groups and readily cross-linked to cell surface components on rat glioma C6 cells which are GM1-deficient. Attachment of the GM1-oligosaccharide derivative, which was monitored by increased binding of 125I-cholera toxin to the cells, was both time- and concentration-dependent. Prior treatment of the cells with dithiothreitol enhanced the attachment by generating additional free sulfhydryl groups. The affinity of cholera toxin for cells treated with the GM1-oligosaccharide derivative or with GM1 was similar. The nature of the newly generated toxin receptors was determined by Western blotting. Membranes from derivatized cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved components were electrophoretically transferred to a nitrocellulose sheet which was overlain with 125I-cholera toxin. The toxin bound to a wide variety of membrane proteins, most of which were trypsin-sensitive. No such binding was observed using membranes from control cells. Although the GM1-neoganglioproteins newly generated on the surface of rat glioma C6 cells readily bound cholera toxin, the cells did not become more responsive to the toxin as measured by increased production of cyclic AMP or activation of adenylate cyclase. In contrast, cells exposed to GM1 became highly responsive to the toxin. Thus, neoganglioproteins on the cell surface appear to behave as nonfunctional receptors for cholera toxin.     

Human platelets are defective in processing of cholera toxin.

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.     

Orientation of cholera toxin bound to model membranes.

The orientation of cholera toxin bound to its cell-surface receptor, ganglioside GM1, in a supporting lipid membrane was determined by electron microscopy of negatively stained toxin-lipid samples. Image analysis of two dimensional crystalline arrays has shown previously that the B-subunits of cholera toxin orient at the membrane surface as a pentameric ring with a central channel (Reed, R. A., J. Mattai, and G.G. Shipley. 1987. Biochemistry. 26:824-832; Ribi, H. O., D. S. Ludwig, K. L. Mercer, G. K. Schoolnik, and R. D. Kornberg. 1988. Science (Wash, DC). 239:1272-1276). We recorded images of negatively stained cholera toxin and isolated B-pentamers oriented perpendicular to the lipid surface so that the pentamer ring is viewed from the side. The pentamer dimensions, estimated from the average of 100 molecules, are approximately 60 by 30 A. Images of side views of whole cholera toxin clearly show density above the pentamer ring away from the lipid layer. On the basis of difference maps between averages of side views of whole toxin and B-pentamers, this density above the pentamer has been identified as a portion of the A-subunit. The A-subunit may also extend into the pore of the pentamer. In addition, Fab fragments from a monoclonal antibody to the A-subunit were mixed with the toxin prior to binding to GM1. Density from the Fab was localized to the region of toxin above the pentamer ring confirming the location of the A-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of growth and induction of enzyme activities in a clonal human hepatoma cell line (Li-7A): comparison of the effects of epidermal growth factor and an anti-epidermal growth factor receptor antibody

A cloned human hepatoma cell line (Li-7A), possessing epidermal growth factor (EGF) receptors numbering in the range of 10-20 pmol/10(6) cells, was inhibited in its growth by EGF as well as an antagonist monoclonal antibody (MoAb) to the EGF receptor. The mode of action of the two ligands of EGF receptors appeared to be different as indicated by the following results: 1) EGF induced marked alteration in cell morphology, whereas the antibody did not; 2) cellular protein accumulated in the EGF-treated cells but not in the antibody treated cells; and 3) ectoATPase activities were greatly enhanced in Li-7A cells treated with EGF and cholera toxin but were unaffected in cells treated with antibody and cholera toxin. The last result also suggests that expression of ectoATPase activities is under the regulation of both EGF and cholera toxin. Li-7A cells provide an additional valuable experimental system for the study of EGF action, as well as the interactive effects of EGF and cholera toxin. The enrichment of the ATPase activities in the EGF-cholera toxin-treated cells can be exploited for the detailed study and isolation of these enzymes and elucidation of their physiological functions.     

Interleukin 3-dependent mouse mast cells express the cholera toxin-binding acidic glycosphingolipid, ganglioside GM1, and increase their histamine content in response to toxin

The acidic glycosphingolipid, ganglioside GM1, which is the binding site for cholera toxin on many cell types, was identified by chemical and by flow cytometric analyses of mouse interleukin 3-dependent, bone marrow culture-derived mast cells (BMMC). Ganglioside GM1 and other acidic glycosphingolipids were isolated from BMMC by chloroform/methanol extraction and chromatography on DEAE-Sephadex and were analyzed by thin layer chromatography. The presence of ganglioside GM1 in the BMMC extract was demonstrated by its co-migration with ganglioside GM1 standard in thin layer chromatography and by the binding of peroxidase-labeled cholera toxin B subunit to both molecules. As assessed by fluorescence flow cytometric analysis of the binding of fluorescein-conjugated cholera toxin B subunit, the majority of BMMC expressed ganglioside GM1 on their surface, and the total presentation per cell increased as cells progressed from the G1 to S to G2 + M phases of the cell cycle. The addition of increasing amounts of cholera toxin starting with 0.08 microgram/ml to BMMC cultured in 50% WEHI 3-conditioned medium containing IL 3 for 48 hr caused the adhesion of BMMC to the tissue culture flasks to increase in a dose-related manner, from less than 1% adherent cells in cultures without toxin to a plateau value of approximately 17% adherent in the presence of 1.25 micrograms/ml of toxin. The histamine content of BMMC increased from 26.7 +/- 3.59 ng/10(6) cells (mean +/- SD, n = 4) for control cultures to 201 +/- 17.4 ng/10(6) cells (mean +/- SD, n = 4) for nonadherent cells and to 588 +/- 89.4 ng/10(6) cells (mean +/- SD, n = 4) for adherent cells after 48 hr of culture in 0.31 microgram/ml cholera toxin, which was the optimal dose for nonadherent and adherent populations. The content of another preformed intragranular mediator, beta-hexosaminidase, did not increase appreciably in the presence of cholera toxin (n = 3). The increase in the histamine content of BMMC after the addition of 0.31 microgram/ml cholera toxin was detectable at 4 hr, plateaued by 24 to 48 hr, and gradually declined over the next 6 days. Cholera toxin also augmented the histamine content of BMMC in the presence of purified synthetic IL 3. Preincubation of whole cholera toxin with purified ganglioside GM1 inhibited the histamine-augmenting effects of cholera toxin on BMMC, indicating that the effect was not due to a contaminant, and neither the A nor B subunit of cholera toxin alone increased the histamine content of BMMC.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ligand-induced formation of the leukotriene B4 receptor-G protein complex of human polymorphonuclear leukocytes.

The components of the polymorphonuclear leukocyte (PMNL) receptor for leukotriene B4 (LTB4) were examined by Sephacryl S-300 exclusion chromatography of PMNL membrane proteins, which were solubilized before and after the binding of [3H] LTB4. When the PMNL membranes were solubilized in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and filtered on Sephacryl S-300 prior to addition of [3H] LTB4, the binding activity was associated with a 65 kD protein. In contrast, the radioactivity of [3H] LTB4 bound to PMNL membranes prior to solubilization was recovered predominantly with a 140 kD protein. When PMNL membranes had been pretreated with pertussis toxin, but not cholera toxin, before the addition of LTB4 and subsequent solubilization, radioactivity was recovered predominantly with the 65 kD protein. The addition of guanylylimidodiphosphate (GMP-PNP), a nonhydrolyzable derivative of guanosine triphosphate (GTP), to PMNL membrane receptors bearing [3H] LTB4 either prior to or after CHAPS solubilization reduced the yield of the 140 kD presumed LTB4 receptor protein-G protein complex. That the maximum specific binding of [35S] guanosine-5'-0-3-thiotriphosphate (GTP-gammaS) to LTB4-binding proteins in the Sephacryl S-300 effluent corresponded to the 140 kD protein supported the presence of a G protein in the LTB4 receptor complex.     

Comparison of the interaction of mono- and oligovalent ligands with cholera toxin. Demonstration of aggregate formation at low ligand concentrations.

The stimulation by cholera toxin of adenylate cyclase in Chinese hamster ovarian cells could be inhibited by various ligands. The latter have been shown to contain the structural oligosaccharide entities required for binding to cholera toxin, established as Galbeta1 leads to 3GalNAcbeta1 leads to 4Gal3 comes from 2alphaNeuAc. The different inhibitory potency of the ligands thereby correlates with the size of the aggregates formed with the toxin, which in turn depends on the valency of the ligands. The conclusion is drawn from a comparison of the interaction of cholera toxin and its B-protomer with ganglioside II3NeuAc-GgOse4-Cer, the newly synthesized bis-(monosialo-gangliotetraityl)amine and monosialogangliotetraose. In a double diffusion test cholera toxin B-protomer precipitated with the ganglioside II3 NeuAcGgOSE4-Cer and the divalent ligand bis(monosialo-gangliotetraityl)amine, suggesting the formation of high molecular weight aggregates, whereas no precipitation was observed with the monovalent monosialo-gangliotetraose. By ultracentrifugation analysis, aggregate formation of the cholera toxin B-protomer could be demonstrated with the ganglioside II3 NeuAc-GgOse4-Cer and bis(monosialo-gangliotetraityl)amine at a concentration at which the ganglioside was assumed to be monodisperse. Ganglioside/cholera toxin B-protomer complexes sediment faster than those of the toxin and bis(monosialo-gangliotetraityl)amine, suggesting higher aggregation of cholera toxin B-protomer with the former. On the other hand, no sedimentation with monosialo-gangliotetraose was observed. By equilibrium displacement dialysis, however, a comparable high affinity of binding to cholera toxin B-protomer of both the mono- and divalent oligosaccharides was demonstrated. Furthermore, values for the maximal concentration of the bound ligand from these binding experiments with cholera toxin B-protomer established molar ratios of ligand to protein of 4 to 1 and 2 to 1 for monosialo-gangliotetraose and bis(monosialo-gangliotetraityl)amine, respectively. From the results it is concluded that the lipophilic moiety of the ganglioside is not directly involved in the binding process to the toxin protein but leads to an oligovalency of this ligand, due to formation of micellar or submicellar structures.     

Synthesis of a photoreactive, radiolabelled derivative of the oligosaccharide of GM1 ganglioside.

A photoreactive, radioiodinatable derivative of the oligosaccharide (GM1OS) of ganglioside GM1 was synthesized as follows: GM1OS was generated from GM1 by ozonolysis and alkaline fragmentation, and reductively aminated to GM1OSNH2 (1-amino-1-deoxymonosialogangliotetraitol). The latter compound was then reacted with N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA) to form GM1OSNH-ASA [1-(4-azidosalicoylamido)-1-deoxymonosialogangliotetraitol], which was radioiodinated and further purified. To test the [125I]GM1OSNH-IASA [1-(4-iodoazidosalicoylamido)-1-deoxymonosialogangliotetraitol+ ++] as a probe for ganglioside-binding proteins, the derivative was incubated with cholera toxin, which specifically binds GM1, followed by photolysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The probe only labelled the B or binding subunit of cholera toxin, but not the A or adenylyl cyclase activating subunit. Labelling was inhibited by excess GM1OS, but not by the oligosaccharides from gangliosides GD1a and GD1b. [125I]GM1OSNH-IASA and analogous oligosaccharide derivatives may be valuable probes for detecting ganglioside-binding proteins.     

Rotavirus virus-like particles administered mucosally induce protective immunity.

We have evaluated the immunogenicity and protective efficacy of rotavirus subunit vaccines administered by mucosal routes. Virus-like particles (VLPs) produced by self-assembly of individual rotavirus structural proteins coexpressed by baculovirus recombinants in insect cells were the subunit vaccine tested. We first compared the immunogenicities and protective efficacies of VLPs containing VP2 and VP6 (2/6-VLPs) and G3 2/6/7-VLPs mixed with cholera toxin and administered by oral and intranasal routes in the adult mouse model of rotavirus infection. VLPs administered orally induced serum antibody and intestinal immunoglobulin A (IgA) and IgG. The highest oral dose (100 microg) of VLPs induced protection from rotavirus challenge (> or = 50% reduction in virus shedding) in 50% of the mice. VLPs administered intranasally induced higher serum and intestinal antibody responses than VLPs administered orally. All mice receiving VLPs intranasally were protected from challenge; no virus was shed after challenge. Since there was no difference in immunogenicity or protective efficacy between 2/6- and 2/6/7-VLPs, protection was achieved without inclusion of the neutralization antigens VP7 and VP4. We also tested the immunogenicities and protective efficacies of 2/6-VLPs administered intranasally without the addition of cholera toxin. 2/6-VLPs administered intranasally without cholera toxin induced lower serum and intestinal antibody titers than 2/6-VLPs administered with cholera toxin. The highest dose (100 microg) of 2/6-VLPs administered intranasally without cholera toxin resulted in a mean reduction in shedding of 38%. When cholera toxin was added, higher levels of protection were achieved with 10-fold less immunogen. VLPs administered mucosally offer a promising, safe, nonreplicating vaccine for rotavirus.     

Hexose transport stimulation and membrane redistribution of glucose transporter isoforms in response to cholera toxin, dibutyryl cyclic AMP, and insulin in 3T3-L1 adipocytes

Exposure of 3T3-L1 adipocytes to 100 ng/ml of cholera toxin or 1 mM dibutyryl cyclic AMP caused a marked stimulation of deoxyglucose transport. A maximal increase of 10- to 15-fold was observed after 12-24 h of exposure, while 100 nM insulin elicited an increase of similar magnitude within 30 min. A short term exposure (4 h) of cells to cholera toxin or dibutyryl cyclic AMP resulted in a 3- to 4-fold increase in deoxyglucose transport which was associated with significant redistribution of both the HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) glucose transporters from low density microsomes to the plasma membrane fraction. Total cellular amounts of both transporter proteins remained constant. In contrast, cells exposed to cholera toxin or dibutyryl cyclic AMP for 12 h exhibited elevations in total cellular contents of GLUT1 (but not GLUT4) protein to about 1.5- and 2.5-fold above controls, respectively. Although such treatments of cells with cholera toxin (12 h) versus insulin (30 min) caused similar 10-fold enhancements of deoxyglucose transport, a striking discrepancy was observed with respect to the content of glucose transporter proteins in the plasma membrane fraction. While insulin elicited a 2.6-fold increase in the levels of GLUT4 protein in the plasma membrane fraction, cholera toxin increased the amount of this transporter by only 30%. Insulin or cholera toxin increased the levels of GLUT1 protein in the plasma membrane fraction equally (1.6-fold). Thus, a greater number of glucose transporters in the plasma membrane fraction is associated with transport stimulation by insulin compared to cholera toxin. We conclude that: 1) at early times (4 h) after the addition of cholera toxin or dibutyryl cyclic AMP to 3T3-L1 adipocytes, redistribution of glucose transporters to the plasma membrane appears to contribute to elevated deoxyglucose uptake rates, and 2) the stimulation of hexose uptake after prolonged treatment (12-18 h) of cells with cholera toxin may involve an additional increase in the intrinsic activity of one or both glucose transporter isoforms.     

Use of magnetic beads in selection and detection of biotoxin aptamers by electrochemiluminescence and enzymatic methods.

Systematic evolution of ligands by exponential enrichment (SELEX) was used to develop DNA ligands (aptamers) to cholera whole toxin and staphylococcal enterotoxin B (SEB). Affinity selection of aptamers was accomplished by conjugating the biotoxins to tosyl-activated magnetic beads. The use of magnetic beads reduces the volumes needed to perform aptamer selection, thus obviating alcohol precipitation and allowing direct PCR amplification from the bead surface. Following five rounds of SELEX, 5'-biotinylated aptamers were bound to streptavidin-coated magnetic beads and used for the detection of ruthenium trisbypyridine [Ru(bpy)3(2+)]-labeled cholera toxin and SEB by an electrochemiluminescence methodology. A comparison of control (double-stranded) aptamer binding was made with aptamers that were heat denatured at 96 degrees C (single-stranded) and allowed to cool (conform) in the presence of biotoxin-conjugated magnetic beads. Results suggest that control aptamers performed equally well when compared to heat-denatured DNA aptamers in the cholera toxin electrochemiluminescence assay and a colorimetric microplate assay employing peroxidase-labeled cholera toxin and 5'-amino terminated aptamers conjugated to N-oxysuccinimide-activated microtiter wells. Interestingly, however, in the SEB electrochemiluminescence assay, double-stranded aptamers exceeded the performance of single-stranded aptamers. The detection limits of all aptamer assays were in the low nanogram to low picogram ranges.     

Comparison of the glycolipid-binding specificities of cholera toxin and porcineEscherichia coli heat-labile enterotoxin: identification of a receptor-active non-ganglioside glycolipid for the heat-labile toxin in infant rabbit small intestine

The binding specificities of cholera toxin andEscherichia coli heat-labile enterotoxin were investigated by binding of¹²⁵I-labelled toxins to reference glycosphingolipids separated on thin-layer chromatograms and coated in microtitre wells. The binding of cholera toxin was restricted to the GM1 ganglioside. The heat-labile toxin showed the highest affinity for GM1 but also bound, though less strongly, to the GM2, GD2 and GD1b gangliosides and to the non-acid glycosphingolipids gangliotetraosylceramide and lactoneotetraosylceramide. The infant rabbit small intestine, a model system for diarrhoea induced by the toxins, was shown to contain two receptor-active glycosphingolipids for the heat-labile toxin, GM1 ganglioside and lactoneotetraosylceramide, whereas only the GM1 ganglioside was receptor-active for cholera toxin. Preliminary evidence was obtained, indicating that epithelial cells of human small intestine also contain lactoneotetraosylceramide and similar sequences. By computer-based molecular modelling, lactoneotetraosylceramide was docked into the active site of the heat-labile toxin, using the known crystal structure of the toxin in complex with lactose. Interactions which may explain the relatively high toxin affinity for this receptor were found.Abbreviations CT cholera toxin - CT-B B-subunits of cholera toxin - LT Escherichia coli heat-labile enterotoxin - hLT humanEscherichia coli heat-labile enterotoxin - pLT porcineEscherichia coli heat-labile enterotoxin - EI electron ionization     

Characterization of the receptor for cholera toxin and Escherichia coli heat-labile toxin in rabbit intestinal brush borders.

125I-labelled heat-labile toxin (from Escherichia coli) and 125I-labelled cholera toxin bound to immobilized ganglioside GM1 and Balb/c 3T3 cell membranes with identical specificities, i.e. each toxin inhibited binding of the other. Binding of both toxins to Balb/c 3T3 cell membranes was saturable, with 50% of maximal binding occurring at 0.3 nM for cholera toxin and 1.1 nM for heat-labile toxin, and the number of sites for each toxin was similar. The results suggest that both toxins recognize the same receptor, namely ganglioside GM1. In contrast, binding of 125I-heat-labile toxin to rabbit intestinal brush borders at 0 degree C was not inhibited by cholera toxin, although heat-labile toxin inhibited 125I-cholera toxin binding. In addition, there were 3-10-fold more binding sites for heat-labile toxin than for cholera toxin. At 37 degrees C cholera toxin, but more particularly its B-subunit, did significantly inhibit 125I-heat-labile toxin binding. Binding of 125I-cholera toxin was saturable, with 50% maximal of binding occurring at 1-2 nM, and was quantitatively inhibited by 10(-8) M unlabelled toxin or B-subunit. By contrast, binding of 125I-heat-labile toxin was non-saturable (up to 5 nM), and 2 X 10(-7) M unlabelled B-subunit was required to quantitatively inhibit binding. Neuraminidase treatment of brush borders increased 125I-cholera toxin but not heat-labile toxin binding. Extensive digestion of membranes with Streptomyces griseus proteinase or papain did not decrease the binding of either toxin. The additional binding sites for heat-labile toxin are not gangliosides. Thin-layer chromatograms of gangliosides which were overlayed with 125I-labelled toxins showed that binding of both toxins was largely restricted to ganglioside GM1. However, 125I-heat-labile toxin was able to bind to brush-border galactoproteins resolved by SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose.     

Receptor-specific large-scale purification of cholera toxin on silica beads derivatized with lysoGM1 ganglioside

1. A receptor-specific affinity chromatographic method for large-scale purification of cholera toxin is described. The receptor ganglioside for cholera toxin, GM1, is hydrolysed to lysoGM1 which is then covalently coupled, via stabilized Schiff's bases, to porous silica beads (Spherosil) onto which a layer of DEAE-dextran has been adsorbed and cross-linked before coupling. Columns of these Spherosil-DEAE-dextran-lysoGM1 beads, in contrast to particles derivatized with lysoGA1, bound the cholera toxin of Vibrio cholerae culture filtrates, after which the toxin could be eluted with the aid of an acid citrate buffer (pH 2.8). 2. The toxin-binding capacity was directly proportional to the amount of lysoGM1 in the column: 2.3 mg/mu mol lysoGM1. The yield of purified toxin after acid elution and pH neutralization was essentially quantitative (83-107%). 3. The affinity-purified toxin contained less than 5% impurities, but consisted of a mixture of predominantly intact holotoxin and B subunit protomer which could readily be separated by gel filtration on Sephadex G-100. 4. Scaling up of the technique was possible: a 1 kg column enabled us to treat 1000-1 cultures of V. cholerae and thus to isolate 20 g of cholera toxin per cycle.     

霍乱疫苗中残余霍乱毒素检测方法的建立及验证

采用间接酶联免疫法,即用神经节苷脂包被,加入待检样品,再加入兔抗霍乱毒素B亚单位抗体,用标准样品的吸光值(A值)对标准样品的浓度绘制4-参数拟合曲线,根据标准曲线计算出待测样品中的CT浓度。结果显示,在浓度范围(0.6～16)ng/ml之间,CT标准浓度和检测浓度成线性关系,r2=0.9986。精确度在浓度范围(0.6～16)ng/ml,CT的平均回收率在96.24%～114.44%之间。精密度:批内变异CV%≤12.98%,批间变异CV%≤18.48%。特异性CT浓度在10ng/ml时,平均回收率为102.6%;CT浓度在5ng/ml时,平均回收率为111.17%;CT浓度在2.5ng/ml时,平均回收率为123.83%。实验表明该方法可检测霍乱疫苗原液中CT的含量。     

Monoclonal antibody-based immunoassay for type A Clostridium botulinum toxin is comparable to the mouse bioassay

A monoclonal antibody (BA11) has been produced against Clostridium botulinum type A neurotoxin by the fusion of myeloma cells (P3 NS1/1-Ag4-1) with spleen cells from BALB/c mice immunized with botulinum type A neurotoxoid. The antibody bound specifically to botulinum type A neurotoxin, showing no cross-reactivity with types B and E botulinum toxins or with any of several other bacterial toxins tested. The monoclonal antibody did not bind to botulinum type A neurotoxin which had been denatured with sodium dodecyl sulfate and bound only weakly to each of the separated heavy and light subunits of the neurotoxin, suggesting a conformational requirement for the antigenic determinant of the antibody. A sensitive immunoassay for C. botulinum type A toxin with monoclonal antibody BA11 in conjunction with an enzyme amplication system has been developed which allows detection of 5 to 10 mouse 50% lethal doses ml-1 of purified neurotoxin. The assay was equally sensitive when applied to the detection of crude toxin in food stuffs; the average value for the minimum level of detectable toxin in extracts of tinned salmon or corned beef was 9 +/- 3.1 mouse 50% lethal doses ml-1.     

Monoclonal antibody-based immunoassay for type A Clostridium botulinum toxin is comparable to the mouse bioassay.

A monoclonal antibody (BA11) has been produced against Clostridium botulinum type A neurotoxin by the fusion of myeloma cells (P3 NS1/1-Ag4-1) with spleen cells from BALB/c mice immunized with botulinum type A neurotoxoid. The antibody bound specifically to botulinum type A neurotoxin, showing no cross-reactivity with types B and E botulinum toxins or with any of several other bacterial toxins tested. The monoclonal antibody did not bind to botulinum type A neurotoxin which had been denatured with sodium dodecyl sulfate and bound only weakly to each of the separated heavy and light subunits of the neurotoxin, suggesting a conformational requirement for the antigenic determinant of the antibody. A sensitive immunoassay for C. botulinum type A toxin with monoclonal antibody BA11 in conjunction with an enzyme amplication system has been developed which allows detection of 5 to 10 mouse 50% lethal doses ml-1 of purified neurotoxin. The assay was equally sensitive when applied to the detection of crude toxin in food stuffs; the average value for the minimum level of detectable toxin in extracts of tinned salmon or corned beef was 9 +/- 3.1 mouse 50% lethal doses ml-1.     

Isolation of cholera toxin receptors from a mouse fibroblast and lymphoid cell line by immune precipitation

Cholera toxin receptors have been isolated from both a mouse fibroblast (Balbc/3T3) and mouse lymphoid cell line labeled by the galactose oxidase borotritiide technique. Tritiated receptor-toxin complexes solubilized in NP40 were isolated by addition of toxin antibody followed by a protein A-containing strain of Staphylococcus aureus. In both cell types by far the major species of toxin receptor isolated was ganglioside in nature, although galactoproteins were also present in the immune complexes. Whether the galactoproteins form part of a toxin-receptor complex or are artifacts of the isolation procedure is presently unclear. The relative specificity of cholera toxin for a carbohydrate sequence in a glycolipid suggests that the toxin might prove a useful tool in establishing the function and organization of glycolipids in membranes. For example, interaction of cholera toxin with the mouse lymphoid cell line was shown to result in patching and capping of bound toxin, raising the possibility that the glycolipid receptor interacts indirectly with cytoskeletal elements. Cholera toxin might also be used to select for mutant fibroblasts lacking the toxin receptor and therefore having an altered glycolipid profile. Such mutants might prove useful in establishing the relationship (if any) between modified glycolipid pattern and other aspects of the transformed phenotype. Attempts to isolate mutants, based on the expectation that growth of cells containing the toxin receptor would be inhibited by the increase in cAMP levels normally induced by cholera toxin, proved unsuccessful. Cholera toxin failed to inhibit significantly the growth of either Balbc or Swiss 3T3 mouse fibroblasts although it markedly elevated cAMP levels.     

Two-Dimensional nmr studies of the interactions between a peptide of cholera toxin and monoclonal antibodies

To increase our understanding of the molecular basis for antibody specificity and for the cross-reactivity of antipeptide antibodies with native proteins, it is important to study the three-dimensional structure of antibody complexes with their peptide antigens. For this purpose it may not be necessary to solve the structure of the whole antibody complex but rather to concentrate on elucidating the combining site structure, the interactions of the antibody with its antigen, and the bound peptide conformation. To extract the information about antibody–peptide interactions and intramolecular interactions in the bound ligand from the complicated and unresolved spectrum of the Fab–peptide complex (Fab: antibody fragment made of Fv—the antibody fragment composed of the variable regions of the light and heavy chains forming a single combining site for the antigen—the light chain, and the first heavy chain constant regions), an nmr methodology based on measurements of two-dimensional transferred nuclear Overhauser effect (NOE) difference spectra was developed. Using this methodology the interactions of three monoclonal antibodies with a cholera toxin peptide were studied. The observed interactions were assigned to the antibody protons involved by specific deuteration of aromatic amino acids and specific chain labeling, and by using a predicted model for the structure of the antibody combining site. The assigned NOE interactions were translated to restraints on interproton distances in the complex that were used to dock the peptide into calculated models for the antibodies combining sites. Comparison of the interactions of three antibodies against a cholera toxin peptide (CTP3). which differ in their cross-reactivity with the toxin, yields information about the size and conformation of antigenic determinants recognized by the antibodies, the structure of their combining sites, and relationships between antibodies' primary structure and their interactions with peptide antigens. © 1994 John Wiley & Sons, Inc.     

Adenylyl cyclase and G-proteins in Phytomonas

Phytomonas sp. membranes have an adenylyl cyclase activity which is greater in the presence of Mn2+ than with Mg2+. The Mg2+ and Mn2+ activity ratio varies from one membrane preparation to another, suggesting that the adenylyl cyclase has a variable activation state. A[35S]GTP-gamma-S-binding activity with a Kd of 171 nM was detected in Phytomonas membranes. Incubation of these membranes with activated cholera or pertussis toxin and [adenylate 23P]NAD+ led to incorporation of radioactivity into bands of about 40-44 kDa. Crude membranes were electrophoresed on SDS-polyacrylamide gels and analyzed, by Western blotting, with the 9188 anti-alpha[s] antibody and the AS/7 antibody (anti-alpha[i], anti-alpha[i1], and anti-alpha[i2]. These procedures resulted in the identification of polypeptides of approximately 40-44 kDa. Phytomonas adenylyl cyclase could be activated by treatment of membrane preparations with cholera toxin, in the presence of NAD+, while similar treatment with pertussis toxin did not affect this enzyme activity. These studies indicate that in Phytomonas, adenylyl cyclase activity is coupled to an unknown receptor entity through G alpha[s] proteins.