Biomedical subjects

C Lingwood

Publications and source records attributed to C Lingwood.

At least 19 recordsLinked to original sources

PubMed2003

3'Sulfogalactolipid binding specifically inhibits Hsp70 ATPase activity in vitro.

A method for the generation of soluble glycosphingolipid derivatives that retain the receptor activity of the parent (BBRC 257:391-394, Carb Res 335:91-100) was used to investigate the consequence of 3'sulfogalactolipid (SGL) specific binding within the N-terminal ATPase-containing domain of Hsc70. Sulfogalactosyl ceramide (SGC) was deacylated, and the resulting sulfogalactosylsphingosine coupled to an alpha-adamantane or a norbornane rigid hydrophobic frame. The resulting conjugate preferentially partitioned into water, as opposed to organic solvent. In the range of 100-300 microM, these conjugates inhibited the specific binding of bovine brain Hsc70 to immobilized SGLs. A similar dose-related inhibition of bovine brain Hsc70 ATPase activity was seen between 100 and 300 microM adamantylSGC (adaSGC). Adamantyl conjugates of glycolipids not bound by Hsp70s had no effect. Kinetic analysis indicated that adaSGC was a noncompetitive inhibitor of Hsc70 ATPase activity, a special case of mixed inhibition since the K(m) values were not statistically different, 0.89 +/- 0.024 microM to 0.93 +/- 0.038 microM, but the V(max) decreased from 0.20 +/- 0.012 pmol min(-1) microg(-1) to 0.15 +/- 0.016 pmol min(-1) microg(-1). A reproducible 5 min lag was observed prior to ATPase inhibition that could be eliminated by preincubation of adaSGC with Hsc70 or by adding the cochaperone Hdj-1. The dependence of ATPase inhibition on the rate of hydrolysis indicates that adaSGC binding occurs at a specific stage of the ATPase cycle. These studies identify a new mechanism for the regulation of Hsp70 ATPase activity.

Acylation↗

PubMed2003

Synergistic effect of verotoxin and interferon-alpha on erythropoiesis.

Previous studies have shown that the verotoxin receptor globotriaosyl ceramide is involved in interferon-alpha (IFN-alpha) signaling pathways. The results of the present study indicate that verotoxin used in combination with IFN-alpha had a direct cytotoxic effect on erythrocyte development by targeting nucleated erythrocyte precursors. Toxin treatment alone had no significant effect on erythropoiesis. However, treatment with 100 ng/ml of verotoxin (VT) in combination with 100 U/ml of IFN-alpha was highly cytotoxic (>90%) to erythroid cells in human cord blood cultures by day 14 post-treatment. The lack of effect on other hematopoietic cells, and the relatively modest decrease in the number of erythroid colonies formed (28%) as a result of IFN-alpha/VT treatment, indicate that the cytotoxicity was targeted specifically toward cells committed to the erythrocyte lineage. IFN-alpha treatment alone did not result in cytotoxicity or a significant reduction in the number of erythroid colonies formed. However, IFN-alpha treatment did result in an increase in the surface expression of verotoxin receptors as determined by flow cytometry following labeling of cells with VT-FITC. Abnormalities in erythrocyte morphology and anemia are associated with infection by verotoxin-producing Escherichia coli such as serotype O157:H7. These symptoms are frequently attributed to passage of erythrocytes through partially occluded blood vessels following toxin-induced damage to endothelial cells. The present results document a synergistic cytotoxic effect of IFN-alpha and verotoxin on erythropoiesis, which could have relevance to clinical infection with verotoxin-producing bacteria.

Drug Synergism↗

PubMed2001

The aglycone of sulfogalactolipids can alter the sulfate ester substitution position required for hsc70 recognition.

3'-Sulfogalactolipids(SGLs), sulfogalactosyl ceramide (SGC), and sulfogalactoglycerolipid (SGG) bind to the N-terminal ATPase-containing domain of members of the heat shock protein 70 family. We have probed this binding specificity using a series of synthetic positional sulfated or phosphorylated glycolipid analogues, containing either a long-chain bisalkyl hydrocarbon-2-(tetradecyl)hexadecane (B30) or C(18) ceramide (SGC(18)) backbone. By TLC overlay and receptor ELISA, recombinant hsc70 bound ceramide-based glycoconjugates having 3'- or 4'-sulfogalactose glycone moieties and the 4'-sulfogalactose positional isomer conjugated to B30. Hsc70 binding was significantly decreased to the 3'-sulfogalactose conjugated to the long-chain branched alkane. 3'-Sulfoglucose conjugated to B30 was not bound, nor were similarly conjugated di-, tri-, and tetra-sulfated or phosphorylated galactolipids. These results highlight the importance of the position, rather than the number of sulfate esters within the galactose ring. This binding selectivity was shared by the sea urchin hsp70-related sperm receptor. A 3'-SGC-based soluble inhibitor, in which the acyl chain was replaced with an adamantyl group, inhibited binding of hsc70 to both 3'- and 4'-SGC species with an IC(50) of 50 and 75 microM, respectively, indicating a shared sulfogalactose binding site. These studies demonstrate the highly specific nature of hsc70/SGL binding and show, for the first time, that the lipid aglycone can alter the substitution position requirement for glycolipid recognition.

Cerebrosides↗

PubMed2001

The ATPase domain of hsp70 possesses a unique binding specificity for 3'-sulfogalactolipids.

The region(s) of hsp70 critical for sulfogalactolipid (SGL) recognition has been defined through deletion analysis and site-directed mutagenesis. Truncated polymerase chain reaction products of hsp70 generated N-terminal fragments of 43, 35, 29, and 22 kDa. The C terminus substrate-binding domain (28 kDa) was also expressed. The N-terminal ATPase domain (rP43) shared the binding specificity of hsp70, because only sulfogalactosyl ceramide and sulfogalactosyl glycerolipid were recognized by both TLC overlay and RELISA. The C-terminal domain showed no binding. SGL binding of rP29 and rP22 was severely reduced. The loss of SGL binding for rP35 by RELISA but not TLC overlay was considered as a function of receptor presentation. The truncation of rP43 to rP35 demonstrates that residues 318-387 (the base of the ATP binding cleft) are critical for high affinity SGL binding. Mutagenesis showed that Arg(342) and Phe(198) are crucial for this process. SGL binding, mediated by these conserved residues within the ATPase domain of hsp70, implies that this binding specificity is evolutionarily conserved.

Adenosine Triphosphatases↗

PubMed2001

Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent-resistant membranes.

In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to the trans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.

Cell Membrane↗

PubMed2001

MHC class II proteins contain a potential binding site for the verotoxin receptor glycolipid CD77.

Globotriaosyl ceramide or CD77 functions as a cell surface receptor for toxins of the Shiga toxin/verotoxin family and as a marker for germinal center stage B-cells. The B-cell protein CD19 and the interferon-alpha receptor possess verotoxin-like amino acid sequences in their extracellular domains, and CD77 has been shown to function in CD19-mediated adhesion and interferon-induced growth inhibition. The Burkitt's lymphoma cell line, Daudi, is similar to germinal center B-cells in their expression of CD77, CD19 and MHC class II molecules. Using the multiple sequence alignment program, ClustalW, we have identified a verotoxin-like amino acid sequence on the beta-chain of human and murine MHC class II molecules. Binding of CD77 at this site could modulate the peptide-binding properties of these MHC class II molecules. Using Western blot analysis of whole cell extracts, we found that CD77-positive Daudi cells have higher levels of HLA-D proteins than VT500 cells, a Daudi-derived CD77-deficient mutant cell line. In contrast, MHC class II-mediated adhesion and surface expression are similar in the two cell lines. Therefore, CD77 could play a functional or regulatory role in MHC class II-mediated functions specifically relating to antigen presentation by B-cells to T helper cells.

Amino Acid Sequence↗

PubMed2000

Glycosphingolipid depletion in fabry disease lymphoblasts with potent inhibitors of glucosylceramide synthase.

BACKGROUND: Fabry disease is an inherited X-linked disorder resulting in the loss of activity of the lysosomal hydrolase alpha-galactosidase A and causing the clinical manifestations of renal failure, cerebral vascular disease, and myocardial infarction. The phenotypic expression of this disorder is manifest by the accumulation of glycosphingolipids containing alpha-galactosyl linkages, most prominently globotriaosylceramide. METHODS: Based on quantitative structure activity studies, we recently reported two newly designed glucosylceramide synthase inhibitors based on 1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4). These inhibitors, 4'-hydroxy-P4 and ethylenedioxy-P4, were evaluated for their ability to deplete globotriaosylceramide and other glucosylceramide-based lipids in Fabry lymphocytes and were compared with N-butyldeoxynojirimycin, another reported glucosylceramide synthase inhibitor. RESULTS: Concentrations as low as 10 nmol/L of 4'-hydroxy-P4 and ethylenedioxy-P4 resulted in 70 and 80% depletion, respectively, of globotriaosylceramide, with maximal depletion occurring at three days of treatment. There was no impairment of cell growth. In contrast, N-butyldeoxynojirimycin only minimally lowered globotriaosylceramide levels, even at concentrations as high as 10 micromol/L. Globotriaosylceramide depletion was confirmed by the loss of binding of FITC-conjugated verotoxin B subunit to the lymphoblasts. CONCLUSIONS: These findings suggest that selective glucosylceramide synthase inhibitors are highly effective in the depletion of globotriaosylceramide from Fabry cell lines. We suggest that these compounds have potential therapeutic utility in the treatment of Fabry disease.

1-Deoxynojirimycin↗

PubMed1999

Verotoxin induces apoptosis and the complete, rapid, long-term elimination of human astrocytoma xenografts in nude mice.

Verotoxin 1 (VT1) is an E. coli elaborated subunit toxin active only against (tumor) cell lines that express the VT1 receptor, globotriaosyl ceramide-Gb3. Astrocytomas can be highly malignant brain tumors that remain refractory to clinical treatment. Some human astrocytoma cell lines are particularly sensitive to VT1 in vitro. To address whether this represents a feasible approach to the elimination of these tumors in man, human astrocytoma tumor xenografts in nude mice were treated with verotoxin. Following a single low-dose intratumoral injection of VT1, complete regression of a 1-cm-diameter tumor within 10 days was observed in all treated animals, without reoccurrence (up to 60 days). Apoptosis was demonstrated in both tumor and vascular cells within the treated xenograft. Verotoxin binding to tumor cells and blood vessels in sections of primary glioblastoma multiforme was found.

Animals↗

PubMed1998

pH-independent retrograde targeting of glycolipids to the Golgi complex.

A small fraction of the molecules internalized by endocytosis reaches the Golgi complex through a retrograde pathway that is poorly understood. In the present work, we used bacterial toxins to study the retrograde pathway in Vero cells. The recombinant B subunit of verotoxin 1B (VT1B) was labeled with fluorescein to monitor its progress within the cell by confocal microscopy. This toxin, which binds specifically to the glycolipid globotriaosyl ceramide, entered endosomes by both clathrin-dependent and -independent pathways, reaching the Golgi complex. Once internalized, the toxin-receptor complex did not recycle back to the plasma membrane. The kinetics of internalization and the subcellular distribution of VT1B were virtually identical to those of another glycolipid-binding toxin, the B subunit of cholera toxin (CTB). Retrograde transport of VT1B and CTB was unaffected by addition of weak bases in combination with concanamycin, a vacuolar-type ATPase inhibitor. Ratio imaging confirmed that these agents neutralized the luminal pH of the compartments where the toxin was located. Therefore, the retrograde transport of glycolipids differs from that of proteins like furin and TGN38, which require an acidic luminal pH. Additional experiments indicated that the glycolipid receptors of VT1B and CTB are internalized independently and not as part of lipid "rafts" and that internalization is cytochalasin insensitive. We conclude that glycolipids utilize a unique, pH-independent retrograde pathway to reach compartments of the secretory system and that assembly of F-actin is not required for this process.

Animals↗

PubMed1997

Expression and sulfogalactolipid binding specificity of the recombinant testis-specific cognate heat shock protein 70.

Immunofluorescent studies with anti-2A antisera, raised specifically against a synthetic C-terminal peptide of native murine P70, the testes-specific cognate heat shock protein 70, demonstrated that the rat homologue of P70 is expressed on the surface of testicular cells. The murine hsp 70.2 gene, encoding P70, was cloned and expressed in Escherichia coli. The recombinant P70 (rP70) protein with a 6Xhistidine affinity tag at its amino terminus was purified from E. coli via nickel affinity column chromatography. Monoclonal anti-hsp70 antisera and anti-2A antisera cross-reacted with purified rP70. Binding of rP70 was specific for sulfogalactosylceramide (SGC) and sulfogalactosyglycerolipid (SGG). Binding was not inhibited by the sugar, galactose 3'sulfate, nor was binding observed to desulfated derivatives of SGC and SGG, to other negatively charged lipids or other sulfated lipids. Furthermore, rP70 bound to an SGC-column and was eluted only at high salt in combination with high pH. These results show rP70 to possess a specific sulfatide binding site. Since the biochemical properties and immunoreactivity of rP70 are indistinguishable from native P70 and SLIP1 (testicular sulfoglycolipid immobilized protein 1) rP70 can be employed to examine the role of hsp70-mediated sulfatide binding in fertilization.

Animals↗

PubMed1997

Brief heat shock treatment induces a long-lasting alteration in the glycolipid receptor binding specificity and growth rate of Haemophilus influenzae.

After brief heat shock treatment, clinical strains of nontypeable Haemophilus influenzae show a long-lasting change in the binding specificity for glycolipids and a markedly increased growth rate in vitro. Non-heat-shocked H. influenzae specifically binds to phosphatidylethanolamine (PE), gangliotetraosylceramide (Gg4), and gangliotriosylceramide (Gg3) and binds minimally to sulfatoxygalactosylceramide (SGC; also called sulfatide). After a 5-min heat shock at 42 degrees C, strains of H. influenzae showed a marked increase in binding to SGC and acquired the ability to bind to sulfatoxygalactosylglycerol (SGG) in thin-layer chromatography overlays. Additionally, heat-shocked H. influenzae cells showed an increased growth rate (twofold). Increased sulfatide binding and growth rate were retained for approximately 60 generations, after which the heat-shocked organisms reverted to their original glycolipid binding pattern (i.e., PE, Gg3, and Gg4) and growth rate. Such organisms could then be reexposed to heat, and the heat shock phenotype would be reestablished. After exposure of the organisms to brief heat shock, Western blotting of a surface extract of H. influenzae with anti-bovine-brain hsp-70 monoclonal antibody showed an increase in two protein bands at 82 and 60 kDa. This antibody was a potent inhibitor of the binding of heat-shocked H. influenzae to SGC and SGG but had no effect on PE, Gg3, or Gg4 binding in vitro. In contrast, an antibody against an H. influenzae PE-Gg3-Gg4-binding adhesin that was recently identified (J. Busse, E. Hartmann, and C. A. Lingwood, J. Infect. Dis. 175:77-83, 1996) selectively inhibited the organism's binding to PE and Gg3. This indicates that cell surface hsp-70-related heat shock proteins can mediate H. influenzae attachment to sulfoglycolipids following heat shock. We suggest that such increased binding to sulfated glycolipids may be a response to fever following H. influenzae infection in humans.

Antibodies, Blocking↗

PubMed1996

Comparative cytotoxicity of purified Shiga-like toxin-IIe on porcine and bovine aortic endothelial and human colonic adenocarcinoma cells.

Porcine and bovine aortic endothelial cells and human colonic adenocarcinoma cells were compared for their susceptibility to the toxic effect of purified Shiga-like toxin IIe (SLT-IIe), measured by the neutral red cytotoxicity assay. Cytotoxicity correlated with toxin binding as indicated by fluorescence activated cell sorter analysis and with the globotriosylceramide (Gb3) and globotetraosylceramide (Gb4) content of cells determined by high pressure liquid chromatography. One line of porcine aortic endothelial cells was 1400-fold more susceptible than the line of bovine aortic endothelial cells that was tested, but a second line of porcine aortic endothelial cells was highly refractory to SLT-IIe. Human colonic adenocarcinoma cells lacked detectable levels of Gb4 and were least susceptible to SLT-IIe.

Adenocarcinoma↗

PubMed1995

Escherichia coli verotoxin binding to human paediatric glomerular mesangial cells.

Haemolytic uraemic syndrome (HUS) remains the leading cause of acute renal failure in children. Although an Escherichia coli-produced verotoxin (VT) has been implicated in the pathogenesis of HUS, the precise mechanisms of disease are not well defined. We hypothesise that the pathogenesis of renal failure in HUS includes the binding of E. coli VT to the glomerular mesangial cell, with consequent effects on renal function. Using human paediatric mesangial cells, we studied the binding and biological effects of the purified verotoxin VT-1. We isolated, purified and characterised paediatric glomerular mesangial cells. The mesangial cells were characterised by their immunoreactivity with both smooth muscle actin and vimentin antibodies, and lack of immunoreactivity with cytokeratin or factor VIII antibodies. Using an fluorescein isothiocyanate-conjugated VT (10(-7)-10(-8) M), we demonstrated specific binding to the mesangial cell membrane by immunofluorescence microscopy. We also demonstrated a dose-dependent inhibition of mesangial cell mitogenesis at concentrations from 10(-9) to 10(-17) M. Our data demonstrate that VT-1 binds to paediatric human glomerular mesangial cells and this binding results in specific biological actions, including an inhibition of cell mitogenesis.

Bacterial Toxins↗

PubMed1994

Increased globotriaosylceramide on plasma membranes of synchronized familial dysautonomia cells. Verotoxin binding studies.

Familial dysautonomia is an autosomal recessive genetic disease found almost exclusively among Ashkenazi Jews, characterized by deficits in autonomic, sensory, and central functions. Although the gene has been localized to chromosome 9, the biochemical defect remains elusive. We previously reported an increase in globotriaosylceramide in dysautonomic fibroblasts and lymphoblasts, and unusual fibroblast growth patterns suggesting plasma membrane abnormalities. Globotriaosylceramide is a plasma membrane component, and the natural receptor for verotoxin derived from E. coli. In Vero and HeLa cells, which are susceptible to verotoxin, the expression of globotriaosylceramide on the cell surface is maximal at the G1/S boundary of the cell cycle. Measurement of toxin binding at 0 degrees C at this boundary is indicative of the amount of globotriaosylceramide exposed on the cell surface. Above 0 degrees C, verotoxin enters, and is toxic to, the cell. We analyzed verotoxin-globotriaosylceramide interactions in synchronized FD and normal cells at this boundary. 125I-toxin binding was much more marked to lymphoblasts from patients than from controls. When cells were grown in the presence of verotoxin, at 10(-2)-10(-7) micrograms/mL, 70% of dysautonomic lymphoblasts died, compared to 25% of controls. The CD50 was 10 ng/mL for dysautonomic fibroblasts vs 450 for controls. These results may be exploited to create a biological assay to differentiate between FD and normal cells.

Autonomic Nervous System Diseases↗

PubMed1993

Alteration of the glycolipid binding specificity of the pig edema toxin from globotetraosyl to globotriaosyl ceramide alters in vivo tissue targetting and results in a verotoxin 1-like disease in pigs.

All members of the verotoxin (VT) family specifically recognize globo-series glycolipids on the surface of susceptible cells. Those toxins that are associated with human disease, VT1, VT2, and VT2c, bind to globotriaosyl ceramide (Gb3) while VT2e, which is associated with edema disease of swine, binds preferentially to globotetraosyl ceramide (Gb4). We were recently able to identify, using site-directed mutagenesis, amino acids in the binding subunit of these toxins that are important in defining their glycosphingolipid (GSL) binding specificity (Tyrrell, G. J., K. Ramotar, B. Boyd, B. W. Toye, C. A. Lingwood, and J. L. Brunton. 1992. Proc. Natl. Acad. Sci. USA. 89:524). The concomitant mutation of Gln64 and Lys66 in the VT2e binding subunit to the corresponding residues (Glu and Gln, respectively) found in VT2 effectively converted the GSL binding specificity of the mutant toxin from Gb4 to Gb3 in vitro. We now report that the altered carbohydrate recognition of the mutant toxin (termed GT3) has biological significance, resulting in a unique disease after intravascular injection into pigs as compared with classical VT2e-induced edema disease. The tissue localization of radiolabeled GT3 after intravascular injection was elevated in neural tissues compared with VT2e accumulation, while localization of GT3 to the gastrointestinal tract was relatively reduced. Accordingly, the pathological lesions after challenge with GT3 involved gross edema of the cerebrum, cerebellum, and brain stem, while purified VT2e caused hemorrhage and edema of the cerebellum, and submucosa of the stomach and large intestine. In addition, both radiolabeled toxins bound extensively to tissues not directly involved in the pathology of disease. VT2e, unlike GT3 or VT1, bound extensively to red cells, which have high levels of Gb4. The overall tissue distribution of VT2e was thus found to be influenced by regional blood flow to each organ and not solely by the Gb4 levels of these tissues. Conversely, the distribution of GT3 (and VT1), which cleared more rapidly from the circulation, correlated with respective tissue Gb3 levels rather than blood flow. These studies indicate the primary role of carbohydrate binding specificity in determining systemic pathology, suggest that the red cells act as a toxin carrier in edema disease, and indicate that red cell binding does not protect against the pathology of systemic verotoxemia.

Animals↗

PubMed1993

Therapeutics used to alleviate peptic ulcers inhibit H. pylori receptor binding in vitro.

Treatment with bismuth-containing remedies has been long associated with the alleviation of minor gastric ailments. Bismuth salts have a potent antimicrobial activity, and are part of the current standard regime used to treat Helicobacter pylori infection. H. pylori is considered to be the major etiological factor in the development of peptic ulcer disease. Earlier efficacious treatments for peptic ulcer included the oral administration of Tween detergents. We have found that these agents have an inhibitory effect on H. pylori adhesion to the lipid species phosphatidylethanolamine (PE) and gangliotetraosylceramide (Gg4) shown previously to be receptors for H. pylori binding in vitro. H. pylori binding to PE and Gg4 was inhibited after a thirty minute preincubation with different bismuth compounds: bismuth subsalicylate > bismuth subgallate > bismuth carbonate > colloidal bismuth subcitrate > tripotassium dicitrato bismuthate. No inhibitory effect on H. pylori binding was observed when bismuth salts were added directly into the binding assay. No changes in bacterial morphology and motility were observed after the thirty minute incubation. Pretreatment with Tween detergents also inhibited H. pylori receptor binding by up to 80% at concentrations as low as 0.0001%. These results suggest that inhibition of H. pylori/host cell adhesion might play a role in efficacious treatment for this infection.

Anti-Ulcer Agents↗

PubMed1993

Comparison of Helicobacter pylori and attaching-effacing Escherichia coli adhesion to eukaryotic cells.

Adhesion of Helicobacter pylori was reported previously to be morphologically identical to "attaching and effacing" Escherichia coli. Therefore, the aim of the present study was to define the adhesion phenotype of H. pylori LC-11 to HEp-2, KATO-III, HEL, and CHO tissue culture cells. By using both staining of F-actin with fluorescein-labeled phalloidin and ultrastructural analysis, diffuse bacterial adhesion to discrete microvillus-denuded regions of the plasma membrane was observed in each of the infected cell lines. However, strain LC-11 did not induce formation of F-actin adhesion pedestals on the eukaryotic cells. H. pylori was negative by colony blot hybridization with an E. coli attaching and effacing gene probe. Elevations in inositol triphosphates followed infection of HEp-2 cells with H. pylori (405% of control values +/- 147%; P < 0.05). To correlate the observed histopathology with expression of the H. pylori phosphatidylethanolamine receptor, a thin-layer chromatography overlay-binding assay was used to identify receptors in each of the cell lines. H. pylori adhered to eukaryotic cells regardless of the presence (HEp-2, KATO-III, and CHO cells) or absence (HEL cells) of the lipid receptor as detected under the assay conditions. However, in comparison to cell lines that possess the phosphatidylethanolamine receptor, HEL cells demonstrated less quantitative H. pylori binding. These findings suggest that mechanisms distinct from E. coli enteropathogens underlie the adhesion of H. pylori to mucosal surfaces. In addition to the phosphatidylethanolamine H. pylori receptor, another host factor(s) likely mediates the attachment of H. pylori to human eukaryotic cells.

Animals↗

PubMed1992

Verotoxin glycolipid receptors determine the localization of microangiopathic process in rabbits given verotoxin-1.

Infection with verotoxin-producing Escherichia coli has been implicated in the cause of hemolytic-uremic syndrome. Cases of thrombotic thrombocytopenic purpura and verotoxin infections have been also described. In this study we sought to determine the following: (1) whether verotoxin induces microvascular lesions in the rabbit, (2) the organ distribution of such lesions, and (3) the distribution of verotoxin glycolipid receptors in the various organs. Rabbits challenged with verotoxin-1 purified from E. coli O157:H7 had anorexia, lethargia, and limb paralysis; renal function, however, was normal. Central nervous system lesions found included pericellular and perivascular edema, focal hemorrhage, vascular lesions, and severe alterations of Purkinje cells. Histologic changes were also seen in the colon, with mucosal and submucosal edema and hemorrhage, and in the lung, with interstitial fibrosis and focal lymphohistiocitic infiltration. No lesions were detected in kidney, heart, liver, and spleen. Screening of various tissues for the presence of the verotoxin receptors revealed galabiosyl ceramide in the central nervous system and globotriosyl ceramide in the gastrointestinal tract, lung, and spleen. No receptors for verotoxin were found in the heart, liver, and kidney. These results indicate that organ localization of the disease in rabbits is dependent on the distribution of verotoxin receptors.

Administration, Oral↗