Peptides binding to a Gb3 mimic selected from a phage library

Peptides binding to a Gb3 mimic were selected from 12-mer peptide library. The self-assembled monolayer (SAM) of a Gb3 mimic was formed on the gold surface, and biopanning was carried out with the phage display peptide library. After three rounds of biopanning, four individual sequences were obtained from 10 phage clones, and the selected peptides having the specific 7-mer sequence (FHENWPS) showed affinities to the Gb3 mimic as strong as to RCA120. Molecular dynamics calculations suggested that the peptides bound to the Gb3 mimic by hydrophobic interaction and hydrogen bonding formation, and the cooperative interactions played an important role in the recognition. The Stx-1 binding was inhibited by the peptides.     

A globotriaosylceramide (Gb3Cer) mimic peptide isolated from phage display library expressed strong neutralization to Shiga toxins

A Gb3-trisaccharide mimic peptide was selected with biopanning from a phage display library against anti-Gb3 antibody to neutralize Shiga toxins (Stxs). Biopanning was carried out on a microplate immobilized with a Fab fragment of anti-Gb3 antibody and a subtraction procedure screening was applied to enhance specificity. The selected phage clones showed strong affinity to anti-Gb3 antibody and to Stxs. Among these clones, a 9-mer sequence WHWTWLSEY was determined as the strongest Gb3 mimic peptide and chemically synthesized. The peptide bound strongly to Stx-1 and Stx-2, though the binding was inhibited with Gb3Cer. Surface plasmon resonance (SPR) and fluorescent spectroscopy determined that the affinity of the peptide to both Stxs was strong. Neutralization activity was confirmed by in vitro assay with HeLa cells. The Gb3 mimic peptide potentially has great promise for use against Stxs.     

A quartz crystal microbalance method for rapid detection and differentiation of shiga toxins by applying a monoalkyl globobioside as the toxin ligand

A simple globobiosyl (Gb2) ceramide mimic carrying a monoalkyl chain (C18) was applied for a monolayer Langmuir-Blodgett (L-B) technique to detect Shiga toxins (Stxs) by a quartz crystal microbalance (QCM) method. The artificial glycolipid, synthesized from penta-O-acetyl-D-galactopyranose via a conventional glycosidation pathway, was developed at the air-water surface for the formation of the monolayer film. Then, the film was transferred onto a QCM cell surface modified with alkanethiols. Upon the addition of each of Stx-1 and Stx-2, the decrease of frequency reached saturation within 45 min at a few nanogram order per quartz cell. Binding constants (Ka) estimated for each of Stx-1 and Stx-2 showed little difference between the two toxins. On the other hand, in the presence of an artificial acrylamido Gb2 copolymer as a competitive inhibitor, the two toxins showed a large difference in the binding behavior to the L-B monolayer.     

Design of multifunctional peptides expressing both antimicrobial activity and shiga toxin neutralization activity

We have designed novel short peptides expressing both antimicrobial and Shiga-toxin (Stx) neutralization activities by combining nuclear localization signal (NLS) peptides (RIRKKLR, PKKKRKV, and PRRRK) tandemly with globotriaoside (Gb3) mimic peptide (WHWTWL). These fusion peptides exhibited excellent antimicrobial activity against both gram-positive and gram-negative bacteria. A peptide WHWTWLRIRKKLR (Trp-His-Trp-Thr-Trp-Leu-Arg-Ile-Arg-Lys-Lys-Leu-Arg), especially, exhibited about 100 times higher activity than the original NLS peptide. SPR analysis demonstrated that the binding of this peptide to both Stxs was strong: K(d) = 6.6 x 10(-6) to Stx-1 and 6.8 x 10(-6) to Stx-2. The in vitro assay against Stx-1 using HeLa cells showed that this peptide increased the survival rate of HeLa cells against the infection of Stx-1. The peptide has been found to maintain high antimicrobial activity, Stx neutralization activity, and no cytotoxicity at its concentration of 7.8-31.3 microg/mL (4.2-16.7 microM). The present peptide design has a prospect of developing potent multifunctional drugs to destroy proteinaceous toxin-producing bacteria and to simultaneously neutralize the toxins released by bacteriolysis.     

Kinetic analysis of binding between Shiga toxin and receptor glycolipid Gb3Cer by surface plasmon resonance

Shiga toxin (Stx) binds to the receptor glycolipid Gb3Cer on the cell surface and is responsible for hemolytic uremic syndrome. Stx has two isoforms, Stx1 and Stx2, and in clinical settings Stx2 is known to cause more severe symptoms, although the differences between the mechanisms of action of Stx1 and Stx2 are as yet unknown. In this study, the binding modes of these two isoforms to the receptor were investigated with a surface plasmon resonance analyzer to compare differences by real time receptor binding analysis. A sensor chip having a lipophilically modified dextran matrix or quasicrystalline hydrophobic layer was used to immobilize an amphipathic lipid layer that mimics the plasma membrane surface. Dose responsiveness was observed with both isoforms when either the toxin concentration or the Gb3Cer concentration was increased. In addition, this assay was shown to be specific, because neither Stx1 nor Stx2 bound to GM3, but both bound weakly to Gb4Cer. It was also shown that a number of fitting models can be used to analyze the sensorgrams obtained with different concentrations of the toxins, and the "bivalent analyte" model was found to best fit the interaction between Stxs and Gb3Cer. This shows that the interaction between Stxs and Gb3Cer in the lipid bilayer has a multivalent effect. The presence of cholesterol in the lipid bilayer significantly enhanced the binding of Stxs to Gb3Cer, although kinetics were unaffected. The association and dissociation rate constants of Stx1 were larger than those of Stx2: Stx2 binds to the receptor more slowly than Stx1 but, once bound, is difficult to dissociate. The data described herein clearly demonstrate differences between the binding properties of Stx1 and Stx2 and may facilitate understanding of the differences in clinical manifestations caused by these toxins.     

Neutralizing activity of polyvalent Gb3, Gb2 and galacto-trehalose models against Shiga toxins

Shiga toxin (Stx) is one of the most critical factors in the development of hemolytic uremic syndrome and other systemic complications following enterohemorrhagic Escherichia coli (EHEC) infection. Substances neutralizing Stx by interfering with toxin-receptor binding have been explored as therapeutic candidates for EHEC infection. In this study, we examined globotriaosyl (Gb3), galabiosyl (Gb2) and galacto-trehalose, each of which was synthetically conjugated with a polyacrylamide backbone, for Stxneutralizing activity. Galacto-trehalose was designed as a Gb2 mimicking, unnatural Stx-ligand that was expected to show tolerance to enzymatic degradation in vivo. Galacto-trehalose copolymer showed neutralizing activity against Stx-1 but not Stx-2 in a HeLa cell cytotoxicity assay. It was thought that galactotrehalose copolymer could be a lead compound for the treatment of Stx-mediated diseases, although it requires modification to show neutralizing activity to Stx-2. The Gb3 copolymer with high sugar unit density showed stronger neutralizing activity against Stx-2 than those with lower density. However, the density-dependency of the neutralizing activity was less obvious against Stx-1. Intravenous administration of the Gb3 copolymer prevented death in mice lethally infected with Stx-1- and Stx-2-producing E. coli O157:H7. Thus, we demonstrated that the artificial Gb3 copolymer could neutralize Stx-1 and the more clinically relevant Stx-2 in vitro and effectively inhibit Stx toxicity in vivo.     

Self-assembled monolayer of light-harvesting core complexes from photosynthetic bacteria on a gold electrode modified with alkanethiols

Light-harvesting antenna core (LH1-RC) complexes isolated from Rhodoseudomonas palustris were self-assembled on a gold electrode modified with self-assembled monolayers (SAMs) of the alkanethiols NH2(CH2)nSH, n = 2, 6, 8, 11; HOOC(CH2)7SH; and CH3(CH2)7SH, respectively. Adsorption of the LH1-RC complexes on the SAMs depended on the terminating group of the alkanethiols, where the adsoption increased in the following order for the terminating groups: amino groups > carboxylic acid groups > methyl groups. Further, the adsorption on a gold electrode modified with SAMs of NH2(CH2)nSH, n = 2, 6, 8, 11, depended on the methylene chain length, where the adsorption increased with increasing the methylene chain length. The presence of the well-known light-harvesting and reaction center peaks of the near infrared (NIR) absorption spectra of the LH1-RC complexes indicated that these complexes were only fully stable on the SAM gold electrodes modified with the amino group. In the case of modification with the carboxyl group, the complexes were partially stable, while in the presence of the terminal methyl group the complexes were extensively denatured. An efficient photocurrent response of these complexes on the SAMs of NH2(CH2)nSH, n = 2, 6, 8, 11, was observed upon illumination at 880 nm. The photocurrent depended on the methylene chain length (n), where the maximum photocurrent response was observed at n = 6, which corresponds to a distance between the amino terminal group in NH2(CH2)6SH and the gold surface of 1.0 nm.     

Specific egg yolk immunoglobulin as a new preventive approach for Shiga-toxin-mediated diseases

Shiga toxins (Stxs) are involved in the development of severe systemic complications associated with enterohemorrhagic Escherichia coli (EHEC) infection. Various neutralizing agents against Stxs are under investigation for management of EHEC infection. In this study, we immunized chickens with formalin-inactivated Stx-1 or Stx-2, and obtained immunoglobulin Y (IgY) from the egg yolk. Anti-Stx-1 IgY and anti-Stx-2 IgY recognized the corresponding Stx A subunit and polymeric but not monomeric B subunit. Anti-Stx-1 IgY and anti-Stx-2 IgY suppressed the cytotoxicity of Stx-1 and Stx-2 to HeLa 229 cells, without cross-suppressive activity. The suppressive activity of these IgY was abrogated by pre-incubation with the corresponding recombinant B subunit, which suggests that the antibodies directed to the polymeric B subunits were predominantly involved in the suppression. In vivo, the intraperitoneal or intravenous administration of these IgY rescued mice from death caused by intraperitoneal injection of the corresponding toxin at a lethal dose. Moreover, oral administration of anti-Stx-2 IgY reduced the mortality of mice infected intestinally with EHEC O157:H7. Our results therefore suggest that anti-Stx IgY antibodies may be considered as preventive agents for Stx-mediated diseases in EHEC infection.     

A globotriaosylceramide (Gb3Cer) mimic peptide isolated from phage display library expressed strong neutralization to Shiga toxins

A Gb₃-trisaccharide mimic peptide was selected with biopanning from a phage display library against anti-Gb₃ antibody to neutralize Shiga toxins (Stxs). Biopanning was carried out on a microplate immobilized with a Fab fragment of anti-Gb₃ antibody and a subtraction procedure screening was applied to enhance specificity. The selected phage clones showed strong affinity to anti-Gb₃ antibody and to Stxs. Among these clones, a 9-mer sequence WHWTWLSEY was determined as the strongest Gb₃ mimic peptide and chemically synthesized. The peptide bound strongly to Stx-1 and Stx-2, though the binding was inhibited with Gb₃Cer. Surface plasmon resonance (SPR) and fluorescent spectroscopy determined that the affinity of the peptide to both Stxs was strong. Neutralization activity was confirmed by in vitro assay with HeLa cells. The Gb₃ mimic peptide potentially has great promise for use against Stxs.     

Adamantyl globotriaosyl ceramide: a monovalent soluble mimic which inhibits verotoxin binding to its glycolipid receptor

The globotriaosylceramide (Gb3) verotoxin (VT) interaction is one of several examples of glycolipid receptors where the ceramide (or lipid) free oligosaccharides fail to show the expected binding parameters. We present a novel, yet simple strategy to synthesize monovalent, water soluble glycosphingolipid mimics which retain receptor function. Replacing the fatty acid chain with rigid, three dimensional hydrocarbon frames, such as adamantane, gives a novel class of neohydrocarbon glycoconjugates. Such adamantyl conjugates derived from Gb3 showed significantly enhanced solubility in water compared to natural Gb3. Adamantyl-Gb3 showed a thousand fold enhanced inhibitory activity (IC50 = 1 microM) for VT-Gb3 binding as compared to a lipid free Gb3 oligosaccharide derivative, alphaGal1-4betaGal1-4betaGlc1-O-CH2CH(CH2SO2C 4H9)2 (IC50 > 2 mM). This represents a new approach to the generation of antagonists of glycolipid receptors.     

Uncommon membrane distribution of Shiga toxin glycosphingolipid receptors in toxin-sensitive human glomerular microvascular endothelial cells

Membrane microdomain association of the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), the highly and less effective receptors, respectively, for Shiga toxins (Stxs), is assumed as a functional requirement for Stx-mediated cytotoxicity. In a previous study, we demonstrated predominant localization of Stx receptors in cholesterol-enriched membrane microdomains of moderately Stx-sensitive human brain microvascular endothelial cells (HBMECs) by means of detergent-resistant membranes (DRMs). Here we report a different preferential distribution of Stx receptors in non-DRM fractions of human glomerular microvascular endothelial cells (GMVECs), the major targets of Stxs in the human kidney. Full structural characterization of Stx receptors using electrospray ionization (ESI) mass spectrometry revealed Gb3Cer and Gb4Cer lipoforms with ceramide moieties mainly composed of C24:0/C24:1 or C16:0 fatty acid and sphingosine (d18:1) in GMVECs comparable to those previously found in HBMECs. Thin-layer chromatography immunostaining demonstrated an approximately 2-fold higher content of Gb3Cer and a 1.4-fold higher content of Gb4Cer in GMVECs than in HBMECs. However, this does not explain the remarkable higher cytotoxic action of Stx1 and Stx2 toward GMVECs as compared with HBMECs. Our finding opens new questions on the microdomain association of Stx receptors and the functional role of GSLs in the membrane assembly of GMVECs.     

Novel redox surfactants and their interactions with glucose oxidase of Aspergillus niger

A number of novel redox surfactants (based on mixed bipyridine/dipyridylamine complexes of osmium (II) where the dipyridylamine ligands bears a saturated C(8), C(10), C(12), C(14), or C(16) alkyl chain) were synthesized and characterized electrochemically and biochemically as mediators for glucose oxidase (EC 1.1.3.4, GOD) of Aspergillus niger. These compounds exhibited critical micelle concentrations (CMCs) in phosphate-buffered saline solution (pH 7.4) in the range 10(-4) 10 10(-3) M, the value decreasing with increasing chain length. Dependence of a number of properties (speed of mediation, redox potential, denaturing action on the enzyme, adsorption on an electrode surface) on the length of the mediator alkyl chain was observed. The presence of an alkyl chain decreased the rate of mediation relative to otherwise similar nonsurfactant mediators, and the longer alkyl chain, the slower the rate of mediation. For each compound, mediation above the CMC was about tenfold slower than that observed below the CMC. However, for the cases of mediator absorbed on an electrode surface with GOD, longer chains give increased physisorption of mixed micelles of enzyme and mediator. The compounds were incidentally found to inhibit the glucose oxidase activity of GOD in a complex manner; inhibition increased with increasing chain length and the deactivation, for any given compound, was more pronounced below the CMC than above. Glucose oxidase activity assays and study of the action of surfactants and mediators on the fluorescent properties of carboxy-fluorescein-labeled GOD led to the consideration of a model for redox surfactant-GOD interaction where three mechanisms may operate: first, a selective interaction of mediators with the GOD active site; second, a nondenaturing association of short-chain (相似文献   

Fatty acid-dependent globotriaosyl ceramide receptor function in detergent resistant model membranes

Glycosphingolipid (GSL) fatty acid strictly regulates verotoxin 1 (VT1) and the HIV adhesin, gp120 binding to globotriaosyl ceramide within Gb(3)/cholesterol detergent resistant membrane (DRM) vesicle constructs and in Gb(3) water-air interface monolayers in a similar manner. VT2 bound Gb(3)/cholesterol vesicles irrespective of fatty acid composition, but VT1 bound neither C18 nor C20Gb(3)vesicles. C18/C20Gb(3) were dominant negative in mixed Gb(3) fatty acid isoform vesicles, but including C24:1Gb(3) gave maximal binding. VT1 bound C18Gb(3) vesicles after cholesterol removal, but C20Gb(3)vesicles required sphingomyelin in addition for binding. HIV-1gp120 also bound C16, C22, and C24, but neither C18 nor C20Gb(3) vesicles. C18 and C20Gb(3) were, in mixtures without C24:1Gb(3), dominant negative for gp120 vesicle binding. Gp120/VT1bound C18 and C24:1Gb(3) mixtures, although neither isoform bound alone. Monolayer surface pressure measurement showed VT1, but not VT2, bound Gb(3) at cellular DRM surface pressures, and confirmed loss of VT1 and gp120 (but not VT2) specific C18Gb(3) binding. We conclude fatty-acid mediated fluidity within simple model GSL/cholesterol DRM can selectively regulate GSL carbohydrate-ligand binding.     

Ricinus communis agglutinin-mediated agglutination and fusion of glycolipid-containing phospholipid vesicles: effect of carbohydrate head group size, calcium ions, and spermine

The glycolipids galactosylcerebroside (GalCer), lactosylceramide (LacCer), and trihexosylceramide (Gb3) were inserted into phospholipid vesicles, consisting of phosphatidylethanolamine and phosphatidic acid. The extent to which their carbohydrate head groups protruded beyond the vesicle surface and their interference with membrane approach were examined by determining vesicle susceptibility toward type I Ricinus communis agglutinin (RCA1) induced agglutination and Ca2+- and spermine-induced aggregation and fusion either in the presence or in the absence of the lectin. The initial agglutination rates increased in the order GalCer much less than LacCer less than Gb3, while a reversed order was obtained for Ca2+- and spermine-induced aggregation and fusion, indicating an enhanced steric interference on close approach of bilayers with increasing head group size. The lectin-mediated agglutination rates for LacCer- and Gb3-containing vesicles increased by an order of magnitude when Ca2+ was also included in the medium, at a concentration that did not induce aggregation per se. Charge neutralization could not account for this observation as the polyvalent cation spermine did not display this synergistic effect with RCA1. Addition of Ca2+ to preagglutinated vesicles substantially reduced the threshold cation concentration for fusion (micromolar vs. millimolar). Quantitatively, this concentration decreased with decreasing carbohydrate head group size, indicating that the head group protrusion determined the interbilayer distance within the vesicle aggregate. The distinct behavior of Ca2+ vs. spermine on RCA1-induced agglutination on the one hand and fusion on the other indicated that Ca2+ regulates the steric orientation of the carbohydrate head group, which appears to be related to its ability to dehydrate the bilayer. As a result, lectin agglutinability becomes enhanced while fusion will be interrupted as the interbilayer distance increases, the threshold head group size being three carbohydrate residues (Gb3). Finally, GalCer-containing vesicles were not agglutinated by RCA1 at ambient temperature, irrespective of the presence of Ca2+. Above 25 degrees C, RCA1 facilitated Ca2+-induced fusion of the vesicles, which was abolished by the haptenic sugar lactose. Since Gb3- and LacCer-containing vesicles displayed a similar behavior, a temperature-induced alteration in the supporting lipid matrix is suggested, which apparently affects lectin/glycolipid interaction.     

Self-assembled monolayers of rigid thiols

The preparation, structure, properties and applications of self-assembled monolayers (SAMs) of rigid 4-mercapto-biphenyls are briefly reviewed. The rigid character of the biphenyl moiety results in a molecular dipole moment that affects both the adsorption kinetics on gold surfaces, as well as the equilibrium structure of mixed SAMs. Due to repulsive intermolecular interaction, the Langmuir isotherm model does not fit the adsorption kinetics of these biphenyl thiols, and a new Ising model was developed to fit the kinetics data. The equilibrium structures of SAMs and mixed SAMs depend on the polarity of the solution from which they were assembled. Infrared spectroscopy suggests that biphenyl moieties in SAMs on gold have small tilt angles with respect to the surfaces normal. Wetting studies shows that surfaces of these SAMs are stable for months, thus providing stable model surfaces that can be engineered at the molecular level. Such molecular engineering is important for nucleation and growth studies. The morphology of glycine crystals grown on SAM surfaces depends on the structure of the nucleating glycine layer, which, in turn, depends on the H-bonding of these molecules with the SAM surface. Finally, the adhesion of PDMS cross-linked networks to SAM surfaces depends on the concentration of interfacial H-bonding. This non-linear relationship suggests that the polymeric nature of the elastomer results in a collective H-bonding effect.     

A novel soluble analog of the HIV-1 fusion cofactor,globotriaosylceramide (Gb(3)), eliminates the cholesterol requirement for high affinity gp120/Gb(3) interaction

We have analyzed the interaction of adamantyl Gb(3) (adaGb(3)), a semi-synthetic soluble analog of Gb(3), with HIV-1 surface envelope glycoprotein gp120. In this analog, which was orginally designed to inhibit verotoxin binding to its glycolipid receptor, Gb(3), the fatty acid chain is replaced with a rigid globular hydrocarbon frame (adamantane). Despite its solubility, adaGb(3) forms monolayers at an air-water interface. Compression isotherms of such monolayers demonstrated that the adamantane substitution resulted in a larger minimum molecular area and a more rigid, less compressible film than Gb(3). Insertion of gp120 into adaGb(3) monolayers was exponential whereas the gp120/Gb(3) interaction curve was sigmoidal with a lag phase of 40 min. Adding cholesterol into authentic Gb(3) monolayers abrogated the lag phase and increased the initial rate of interaction with gp120. This effect of cholesterol was not observed with phosphatidylcholine or sphingomyelin. In addition, verotoxin-bound adaGb(3) or Gb(3) plus cholesterol was recovered in fractions of comparable low density after ultracentrifugation through sucrose-density gradients in the presence of Triton X-100. The unique biological and physico-chemical properties of adaGb(3) suggest that this analog may be a potent soluble mimic of Gb(3), providing a novel concept for developing GSL-derived viral fusion inhibitors.     

Shiga toxin glycosphingolipid receptors in microvascular and macrovascular endothelial cells: differential association with membrane lipid raft microdomains

Vascular damage caused by Shiga toxin (Stx)-producing Escherichia coli is largely mediated by Stxs, which in particular, injure microvascular endothelial cells in the kidneys and brain. The majority of Stxs preferentially bind to the glycosphingolipid (GSL) globotriaosylceramide (Gb3Cer) and, to a lesser extent, to globotetraosylceramide (Gb4Cer). As clustering of receptor GSLs in lipid rafts is a functional requirement for Stxs, we analyzed the distribution of Gb3Cer and Gb4Cer to membrane microdomains of human brain microvascular endothelial cells (HBMECs) and macrovascular EA.hy 926 endothelial cells by means of anti-Gb3Cer and anti-Gb4Cer antibodies. TLC immunostaining coupled with infrared matrix-assisted laser desorption/ionization (IR-MALDI) mass spectrometry revealed structural details of various lipoforms of Stx receptors and demonstrated their major distribution in detergent-resistant membranes (DRMs) compared with nonDRM fractions of HBMECs and EA.hy 926 cells. A significant preferential partition of different receptor lipoforms carrying C24:0/C24:1 or C16:0 fatty acid and sphingosine to DRMs was not detected in either cell type. Methyl-β-cyclodextrin (MβCD)-mediated cholesterol depletion resulted in only partial destruction of lipid rafts, accompanied by minor loss of GSLs in HBMECs. In contrast, almost entire disintegration of lipid rafts accompanied by roughly complete loss of GSLs was detected in EA.hy 926 cells after removal of cholesterol, indicating more stable microdomains in HBMECs. Our findings provide first evidence for differently stable microdomains in human endothelial cells from different vascular beds and should serve as the basis for further exploring the functional role of lipid raft-associated Stx receptors in different cell types.     

Impact of self-assembly composition on the alternate interfacial electron transfer for electrostatically immobilized cytochrome c

We report on the effects of self-assembled monolayer (SAM) dilution and thickness on the electron transfer (ET) event for cytochrome c (CytC) electrostatically immobilized on carboxyl terminated groups. We observed biphasic kinetic behavior for a logarithmic dependence of the rate constant on the SAM carbon number (ET distance) within the series of mixed SAMs of C(5)COOH/C(2)OH, C(10)COOH/C(6)OH, and C(15)COOH/C(11)OH that is in overall similar to that found earlier for the undiluted SAM assemblies. However, in the case of C(15)COOH/C(11)OH and C(10)COOH/C(6)OH mixed SAMs a notable increase of the ET standard rate constant was observed, in comparison with the corresponding unicomponent (omega-COOH) SAMs. In the case of the C(5)COOH/C(2)OH composite SAM a decrease of the rate constant versus the unicomponent analogue was observed. The value of the reorganization free energy deduced through the Marcus-like data analysis did not change throughout the series; this fact along with the other observations indicates uncomplicated rate-determining unimolecular ET in all cases. Our results are consistent with a model that considers a changeover between the alternate, tunneling and adiabatic intrinsic ET mechanisms. The physical mechanism behind the observed fine kinetic effects in terms of the protein-rigidifying omega-COOH/CytC interactions arising in the case of mixed SAMs are also discussed.     

Characterization of a shiga-toxin 1-resistant stock of vero cells

Shiga toxins (Stxs, also referred to as verotoxins) were first described as a novel cytotoxic activity against Vero cells. In this study, we report the characterization of an Stx1-resistant (R-) stock of Vero cells. (1) When the susceptibility of R-Vero cells to Stx1 cytotoxicity was compared to that of Stx1-sensitive (S-) Vero cells by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, cell viability after 48-hr exposure to 10 pg/ml of Stx1 was greater than 80% and less than 15%, respectively. (2) Although both a binding assay of fluorescence-labeled Stx1 and lipid analysis indicated considerable expression of Gb3Cer, a functional receptor for Stxs, in both Vero cells, anti-Gb3Cer monoclonal antibodies capable of binding to S-Vero cells failed to effectively label R-Vero cells, suggesting a conformational difference in the Gb3Cer expressed on R-Vero cells. (3) The lipid analysis also showed that the R-Vero cells contained significant amounts of Gb4Cer. In addition, introduction of exogenous Gb4Cer into S-Vero cells slightly inhibited Stx1 cytotoxicity, suggesting some correlation between glycosphingolipid composition and Stx1 resistance. (4) Both butyrate treatment and serum depression eliminated the Stx1 resistance of R-Vero cells. (5) The results of the analysis by confocal microscopy suggest a difference in intracellular transport of Stx1 between R-Vero and S-Vero cells. Further study of R-Vero cells may provide a model of Stx1 resistance via distinct intracellular transport of Stx1.