数学模型法研究L．casei LC2W对H.pylori SS1黏附MKN-45细胞的抑制作用

目的研究干酪乳杆菌LC2W对幽门螺杆菌（H.pylori）SS1黏附MKN-45的抑制作用,探讨益生菌对致病菌拈抗的机制。方法体外培养人胃癌细胞MKN-45,采用平板计数的方法研究2株细菌的黏附性质;引入数学模型,比较LC2W与H.pylori SS1的竞争、排除和替代作用。结果运用模型可以估算出LC2W和H.pylori SS1对MKN-45最大黏附数和亲和力的大小,并可以预测在混合体系中2种菌黏附的比例;实验发现LC2W对H.pylori SS1的黏附具有很强的竞争作用和排除作用,且这2种作用存在明显的量效关系。LC2W对H.pylori SS1的黏附的替代作用不明显或过程非常缓慢。结论所采用的数学模型能较好的模拟LC2W和H.pylori SS1黏附及LC2W对H.pylori SS1黏附抑制作用,这种抑制作用主要是通过竞争性占位形成的。     

Combined effect of rebamipide and ecabet sodium on Helicobacter pylori adhesion to gastric epithelial cells

Helicobacter pylori is a major etiological agent in gastroduodenal disorders. The adhesion of H. pylori to gastric epithelial cells is the initial step of H. pylori infection. Inhibition of H. pylori adhesion is thus a therapeutic target in the prevention of H. pylori infection. We have reported that rebamipide and ecabet sodium, mucoprotective antiulcer agents, independently inhibit H. pylori adhesion. However, the antiadhesion activity of each antiulcer agent was incomplete. Experiments were performed to evaluate the combined effect of rebamipide and ecabet sodium on H. pylori adhesion to gastric epithelial cells. MKN-28 and MKN-45 cells, derived from human gastric carcinomas, were used as target cells. Twelve clinical isolates of H. pylori were used in this study. We evaluated the effects of rebamipide and ecabet sodium, individually and in combination, on H. pylori adhesion to target cells quantitatively using our previously established enzyme-linked immunosorbent assay. Rebamipide and ecabet sodium each partially inhibited H. pylori adhesion. In contrast, adhesion was almost completely inhibited by pretreating target cells and H. pylori with the combination of rebamipide and ecabet sodium. Our studies suggest that the synergistic antiadhesion activity of rebamipide and ecabet sodium is greater than that of each antiulcer agent alone.     

Enhanced binding of the neural siglecs, myelin-associated glycoprotein and Schwann cell myelin protein, to Chol-1 (alpha-series) gangliosides and novel sulfated Chol-1 analogs

Extended glycoconjugate binding specificities of three sialic acid-dependent immunoglobulin-like family member lectins (siglecs), myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), and sialoadhesin, were compared by measuring siglec-mediated cell adhesion to immobilized gangliosides. Synthetic gangliosides bearing the alpha-series determinant (NeuAc alpha2,6-linked to GalNAc on a gangliotetraose core) were tested, including GD1alpha (IV(3)NeuAc, III(6)NeuAc-Gg(4)OseCer), GD1alpha with modified sialic acid residues at the III(6)-position, and the "Chol-1" gangliosides GT1aalpha (IV(3)NeuAc, III(6)NeuAc, II(3)NeuAc-Gg(4)OseCer) and GQ1balpha (IV(3)NeuAc, III(6)NeuAc, II(3)(NeuAc)(2)-Gg(4)OseCer). The alpha-series gangliosides displayed enhanced potency for MAG- and SMP-mediated cell adhesion (GQ1balpha > GT1aalpha, GD1alpha > GT1b, GD1a > GM1 (nonbinding)), whereas sialoadhesin-mediated adhesion was comparable with alpha-series and non-alpha-series gangliosides. GD1alpha derivatives with modified sialic acids (7-, 8-, or 9-deoxy) or sulfate (instead of sialic acid) at the III(6)-position supported adhesion comparable with that of GD1alpha. Notably, a novel GT1aalpha analog with sulfates at two internal sites of sialylation (NeuAcalpha2,3Galbeta1,4GalNAc-6-sulfatebeta1, 4Gal3-sulfatebeta1,4Glcbeta1,1'ceramide) was the most potent siglec-binding structure tested to date (10-fold more potent than GT1aalpha in supporting MAG and SMP binding). Together with prior studies, these data indicate that MAG and SMP display an extended structural specificity with a requirement for a terminal alpha2, 3-linked NeuAc and great enhancement by nearby precisely spaced anionic charges.     

Interleukin-2 binds to ganglioside GD(1b)

We have developed a solid matrix immunoassay to determine the binding of interleukin-2 (IL-2) to specific gangliosides. The assay establishes that recombinant human IL-2 binds to ganglioside GD(1b) but not to any other gangliosides (GM(1), GM(2), GM(3), GD(1a), GD(2), GD(3), and GT(1b)). The binding varies with the ratio of GD1b and IL-2. This assay enables distinguishing the nature of the sugar moiety of the ganglioside recognized by IL-2 and establishes the dosimetry of the ganglioside-IL-2 interaction. Since rIL-2 is administered systematically into stage IV melanoma patients, we have examined 45 tumor biopsies for GD(1b) content. The incidence of GD(1b) in tumor biopsies is 51%. We postulate that GD(1b) associated on the tumor or in the circulation of cancer patients may bind to rIL-2 and prevent the availability of rIL-2 to augment antitumor-immune response.     

Regulation of Ca2+/calmodulin-dependent protein kinase II by brain gangliosides

Purified rat brain Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca2+/calmodulin (CaM). Of the various gangliosides tested, GT1b was the most potent, giving half-maximal activation at 25 microM. Gangliosides GD1a and GM1 also gave activation, but asialo-GM1 was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM-kinase II on serine residues, but did not produce the Ca2+-independent form of the kinase. Ganglioside stimulation of CaM-kinase II was also present in rat brain synaptic membrane fractions. Higher concentrations (125-250 microM) of GT1b, GD1a, and GM1 also inhibited CaM-kinase II activity. This inhibition appears to be substrate-directed, as the extent of inhibition is very dependent on the substrate used. The molecular mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 281-309), which contains the CaM-binding, inhibitory, and autophosphorylation domains of CaM-kinase II. Using purified brain CaM-kinase II in which these regulatory domains were removed by limited proteolysis. CaMK 281-309 strongly inhibited kinase activity (IC50 = 0.2 microM). GT1b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine-286. These results demonstrate that GT1b can partially mimic the effects of Ca2+/CaM on native CaM-kinase II and on peptide CaMK 281-309.     

Inhibition of human neuroblastoma cell proliferation and EGF receptor phosphorylation by gangliosides GM1, GM3, GD1A and GT1B

The inhibitory action of gangliosides GT1B, GD1A, GM3 and GM1 on cell proliferation and epidermal growth factor receptor (EGFR) phosphorylation was determined in the N-myc amplified human neuroblastoma cell line NBL-W. The IC50 of each ganglioside was estimated from concentration-response regressions generated by incubating NBL-W cells with incremental concentrations (5-1000 microm) of GT1B, GD1A, GM3 or GM1 for 4 days. Cell proliferation was quantitatively determined by a colourimetric assay using tetrazolium dye and spectrophotometric analysis, and EGFR phosphorylation by densitometry of Western blots. All gangliosides assayed, with the exception of GM1, inhibited NBL-W cell proliferation in a concentration-dependent manner. The IC50s for gangliosides GT1B [molecular weight (MW) 2129], GM3 (MW 1236), and GD1A (MW 1838) were (mean +/- SEM) 117 +/- 26, 255 +/- 29, and 425 +/- 44 m, respectively. In contrast, the IC50 for GM1 (MW 1547) could not be determined. Incubation of NBL-W cells with epidermal growth factor (EGF) concentrations ranging from 0.1 to 1000 ng/ml progressively increased cell proliferation rate, but it plateaued at concentrations above 10 ng/ml. EGFR tyrosine phosphorylation, however, was incrementally stimulated by EGF concentrations from 1 to 100 ng/ml. The suppression of EGF-induced EGFR phosphorylation differed for each ganglioside, and their respective inhibitory potencies were as follows: EGFR phosphorylation [area under curve (+ EGF)/area under curve (- EGF)]: control (no ganglioside added) = 8.2; GM1 = 8.3; GD1A = 6.7; GM3 = 4.87, and GT1B = 4.09. The lower the ratio, the greater the inhibitory activity of the ganglioside. Gangliosides GD1A and GT1B, which have terminal N-acetyl neuraminic acid moieties, as well as one and two N-acetyl neuraminic acid residues linked to the internal galactose, respectively, both inhibited cell proliferation and EGFR phosphorylation. However, GD1A was a more potent suppressor of cell proliferation and GT1B most effective against EGFR phosphorylation. GM3, which only has a terminal N-acetyl neuraminic acid, inhibited cell proliferation and EGFR phosphorylation almost equivalently. These data suggest that gangliosides differ in their potency as inhibitors of NBL-W neuroblastoma cell proliferation and EGFR tyrosine phosphorylation, and that perturbations in the differential expression of membrane glycosphingolipids may play a role in modulating neuroblastoma growth.     

Adhesion of Human Glioma Cell Lines to Fibronectin, Laminin, Vitronectin and Collagen I Is Modulated by Gangliosides in vitro

Adhesion of eight cell lines, derived from human gliomas of different histological types, to fibronectin, collagen I, vitronectin, and laminin was investigated in vitro. The glioma cell lines were found to attach to these substrates to different extents. Interestingly, all cell lines strongly attached to laminin. In addition, glioma cell adhesion was found to be dose dependent. Moreover, adhesion of three cell lines to fibronectin and collagen I was partially inhibited and to vitronectin completely prevented by GRGDTP peptide, indicating the involvement of integrin receptors in glioma cell adhesion. We have demonstrated, recently, that gangliosides play an important role in promoting glioma cell invasion of the reconstituted basement membrane, Matrigel, in vitro. In order to study the mechanism of action of gangliosides in this process, the role of six gangliosides (GM1, GM3, GD3, GD1a, GD1b, and GT1b) in cell adhesion to the four proteins was investigated in three cell lines. Although all gangliosides, with the exception of GM3, were found to enhance cell adhesion to these proteins to different extents, GD3 proved to be the most effective adhesion-promoting ganglioside in all three cell lines. GM3 was found to inhibit cell adhesion to the four proteins in one cell line but enhanced cell adhesion in two other cell lines. The three cell lines were found to express both GD3 and gangliosides recognised by the A2B5 antibody. Furthermore, adhesion of the three cell lines to fibronectin, vitronectin, laminin, and collagen I was inhibited by incubation with A2B5, demonstrating the involvement of intrinsic cell membrane gangliosides in adhesion of glioma cells to these proteins. Taken together with the observation that gangliosides modulate integrin receptor function, these data suggest that gangliosides may play a central role in the control of the adhesive and invasive properties of human glioma cells.     

Fibronectin binding to gangliosides and rat liver plasma membranes

Binding of fibronectins to gangliosides was tested directly using several different in vitro models. Using an enzyme-linked immunoabsorbent assay (ELISA), gangliosides were immobilized on polystyrene tubes and relative binding of fibronectin was estimated by alkaline phosphatase activity of conjugated second antibody. Above a critical ganglioside concentration, the gangliosides bound the fibronectin (GT1b congruent to GD1b congruent to GD1a greater than GM1 much greater than GM2 congruent to GD3 congruent to GM3) in approximately the same order of efficiency as they competed for the cellular sites of fibronectin binding in cell attachment assays (Kleinman et al., Proc natl acad sci US 76 (1979) 3367). Alternatively, these same gangliosides bound to immobilized fibronectin. Rat erythrocytes coated with gangliosides GM1, GD1a or GT1b bound more fibronectin than erythrocytes not supplemented with gangliosides. Using fibronectin in which lysine residues were radioiodinated, an apparent Kd for binding to mixed rat liver gangliosides of 7.8 X 10(-9) M was determined. This value compared favorably with the apparent Kd for attachment of fibronectin to isolated plasma membranes from rat liver of 3.7 X 10(-9) M for fibronectin modified on the tyrosine residue, or 6.4 X 10(-9) M for fibronectin modified on lysine residues. As shown previously by Grinnell & Minter (Biochem biophys acta 550 (1979) 92), fibronectin modified on tyrosine residues did not promote spreading and attachment of CHO cells. It did, however, bind to cells. In contrast, lysine-modified fibronectin both bound to cells and promoted cell attachment. Plasma membranes isolated from hepatic tumors in which the higher gangliosides that bind fibronectin were depleted bound 43-75% less [125I]fibronectin than did plasma membranes from control livers. The findings were consistent with binding of fibronectins to gangliosides, including the same gangliosides depleted from cell surfaces during tumorigenesis in the rat.     

Carbohydrate-carbohydrate binding of ganglioside to integrin alpha(5) modulates alpha(5)beta(1) function

Gangliosides GT1b and GD3, components of keratinocyte membranes, inhibit keratinocyte adhesion to fibronectin. Although ganglioside sialylation is known to be important, the mechanism of inhibition is unknown. Using purified insect recombinant alpha(5) and beta(1) proteins and alpha(5)beta(1) integrin from lysed keratinocyte-derived SCC12 cells, we have shown that GT1b and GD3 inhibit the binding of alpha(5)beta(1) to fibronectin. Co-immunoprecipitation of GT1b and alpha(5)beta(1) from SCC12 cells and direct binding of GT1b and GD3 to affinity-purified alpha(5)beta(1) from SCC12 cells and insect recombinant alpha(5)beta(1), particularly the alpha(5) subunit, further suggest interaction between ganglioside and alpha(5)beta(1). The carbohydrate moieties of integrin appear to be critical since gangliosides are unable to bind deglycosylated forms of alpha(5)beta(1) from SCC12 and insect cells or poorly glycosylated recombinant alpha(5)beta(1) from Escherichia coli cells. The GT1b-alpha(5)beta(1) interaction is inhibited by concanavalin A, suggesting that GT1b binds to mannose structures in alpha(5)beta(1). The preferential binding of GT1b to high mannose rather than reduced mannose ovalbumin further implicates the binding of GT1b to mannose structures. These data provide evidence that highly sialylated gangliosides regulate alpha(5)beta(1)-mediated adhesion of epithelial cells to fibronectin through carbohydrate-carbohydrate interactions between GT1b and the alpha(5) subunit of alpha(5)beta(1) integrin.     

Generation of one set of monoclonal antibodies specific for b-pathway ganglio-series gangliosides.

We established six murine monoclonal antibodies (MAbs) specific for b-pathway ganglio-series gangliosides by immunizing C3H/HeN mice with these purified gangliosides adsorbed to Salmonella minnesota mutant R595. The binding specificities of these MAbs were determined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatogram. These six MAbs, designated GGB19, GMR2, GMR7, GGR12, GMR5, and GGR13 reacted strongly with the gangliosides GD3, O-Ac-GD3, GD2, GD1b, GT1b, and GQ1b, respectively, that were used as immunogens. All these MAbs except GGB19 showed highly restricted binding specificities, reacting only with the immunizing ganglioside. None of other various authentic gangliosides or neutral glycolipids were recognized. On the other hand, MAb GGB19 exhibited a broader specificity, cross-reacting weakly with O-Ac-GD3, GQ1b, and GT1a, but not with other gangliosides or neutral glycolipids. Using these MAbs, we determined the expression of these gangliosides, especially GD1b, GT1b, and GQ1b on mouse, rat, and human leukemia cells. GD1b was expressed on rat leukemia cells, but not on mouse and human leukemia cells tested. Neither GT1b nor GQ1b was detected in these cell lines.     

Campylobacter pylori colonization factor shows specificity for lactosylceramide sulfate and GM3 ganglioside

The specificity of Campylobacter pylori cell surface lectin, a presumptive colonization factor, was investigated using various sulfated and sialic acid containing glycolipids. C. pylori cells, cultured from human antral mucosal biopsies, were incubated with intact and modified glycolipid preparations and examined for agglutination inhibition of human erythrocytes. Titration data revealed that the inhibitory activity was highest with lactosylceramide sulfate and GM3 ganglioside, while galactosylceramide sulfate GM1, GD1a and GD1b gangliosides were less effective. A strong inhibitory activity towards C. pylori hemagglutin was also observed with an antiulcer agent, sucralfate. The inhibitory effect of both types of glycolipids was abolished by the removal of sialic acid and sulfate ester groups, thus indicating that sulfated and sialic acid containing glycolipids with terminal lactosyl moieties serve as mucosal receptors for colonization of gastric epithelium by C. pylori.     

Gangliosides inhibit flagellin signaling in the absence of an effect on flagellin binding to toll-like receptor 5

A recent study (Ogushi, K., Wada, A., Niidome, T., Okuda, T., Llanes, R., Nakayama, M., Nishi, Y., Kurazono, H., Smith, K. D., Aderem, A., Moss, J., and Hirayama, T. (2004) J. Biol. Chem. 279, 12213-12219) concluded that gangliosides serve as co-receptors for flagellin signaling via toll-like receptor 5 (TLR5). In view of several findings in this study that were inconsistent with a role for gangliosides as co-receptors, we re-examined this important issue. Using TLR5-negative RAW 264.7 cells and a TLR5-enhanced yellow fluorescent protein chimera, we established an assay for specific binding of flagellin to cells. Inhibition of clatherin-mediated internalization of flagellin.TLR5-enhanced yellow fluorescent protein complexes did not impair flagellin activation of IRAK-1. Thus flagellin signal occurs at the cell surface and not intracellularly. Exogenous addition of mixed gangliosides (GM1, GD1a, and GT1b) as well as GD1a itself inhibited flagellin-induced interleukin-1 receptor-associated kinase activation as well as tumor necrosis factor alpha production in HeNC2, THP-1, and RAW 264.7 cells. Gangliosides inhibited flagellin signaling in the absence of an effect on flagellin binding to TLR5. Depletion of gangliosides in RAW 264.7 cells did not alter the concentration dependence or magnitude of flagellin signaling as measured by interleukin-1 receptor-associated kinase activation or tumor necrosis factor alpha production. Our findings are consistent with the conclusions that gangliosides are not essential co-receptors for flagellin and that the inhibitory effect of gangliosides is mediated by at least one mechanism that is distinct from any effect on the binding of flagellin to TLR5.     

Regulation of Ca2+/Calmodulin-Dependent Protein Kinase II by Brain Gangliosides

Abstract: Purified rat brain Ca²⁺/calmodulin-dependent protein kinase II (CaM-kinase II) is stimulated by brain gangliosides to a level of about 30% the activity obtained in the presence of Ca²⁺/calmodulin (CaM). Of the various gangliosides tested, GT1b was the most potent, giving half-maximal activation at 25 μ M . Gangliosides GD1a and GM1 also gave activation, but asialo-GM1 was without effect. Activation was rapid and did not require calcium. The same gangliosides also stimulated the autophosphorylation of CaM-kinase II on serine residues, but did not produce the Ca²⁺-independent form of the kinase. Ganglioside stimulation of CaM-kinase II was also present in rat brain synaptic membrane fractions. Higher concentrations (125-250 μ M ) of GT1b, GD1a, and GM1 also inhibited CaM-kinase II activity. This inhibition appears to be substrate-directed, as the extent of inhibition is very dependent on the substrate used. The molecular mechanism of the stimulatory effect of gangliosides was further investigated using a synthetic peptide (CaMK 281-309), which contains the CaM-binding, inhibitory, and autophosphorylation domains of CaM-kinase II. Using purified brain CaM-kinase II in which these regulatory domains were removed by limited proteolysis, CaMK 281-309 strongly inhibited kinase activity (IC₅₀=0.2 μ M ). GT1b completely reversed this inhibition, but did not stimulate phosphorylation of the peptide on threonine-286. These results demonstrate that GT1b can partially mimic the effects of Ca²⁺/CaM on native CaM-kinase II and on peptide CaMK 281-309.     

Immunoglobulin G-class mouse monoclonal antibodies to major brain gangliosides

Mice genetically engineered to lack complex gangliosides are improved hosts for raising antibodies against those gangliosides. We report the generation and characterization of nine immunoglobulin G (IgG)-class monoclonal antibodies (mAbs) raised against the four major brain gangliosides in mammals. These include (designated as ganglioside specificity-IgG subclass) two anti-GM1 mAbs (GM1-1, GM1-2b), three anti-GD1a mAbs (GD1a-1, GD1a-2a, GD1a-2b), one anti-GD1b mAb (GD1b-1), and three anti-GT1b mAbs (GT1b-1, GT1b-2a, GT1b-2b). Each mAb demonstrated high specificity, with little or no cross-reactivity with other major brain gangliosides. Enzyme-linked immunosorbent assay (ELISA) screening against 14 closely related synthetic and purified gangliosides confirmed the high specificity, with no significant cross-reactivity except that of the anti-GD1a mAbs for the closely related minor ganglioside GT1a alpha. All of the mAbs were useful for ELISA, TLC immunooverlay, and immunocytochemistry. Neural cells from wild-type rats and mice were immunostained to differing levels with the anti-ganglioside antibodies, whereas neural cells from mice engineered to lack complex gangliosides (lacking the ganglioside-specific biosynthetic enzyme UDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase) remained unstained, demonstrating that most of the mAbs react only with gangliosides and not with related structures on glycoproteins. These mAbs may provide useful tools for delineation of the expression and function of the major brain gangliosides and for probing the pathology of anti-ganglioside autoimmune diseases.     

Composition of gangliosides from ovine testis and spermatozoa

Gangliosides were extracted and purified from ovine testis and ejaculated spermatozoa which contained, respectively, 57 and 9 nmol lipid-bound sialic acid per gram wet weight. Fourteen gangliosides were resolved by thin-layer chromatography of testicular gangliosides, of which eleven were purified in sufficient quantity to enable a complete compositional analysis of the carbohydrate residues to be performed. None of the gangliosides contained fucose, but several contained N-glycolylneuraminic acid as a component of the sialic acid species. Relative migration on thin-layer chromatograms relative to known standards, compositional analysis, and selective degradation by specific enzymes were used as the basis for identification. Testis contained members of the ganglio series (GM1, GD1a, GD1b, GT1b, GQ1b), hematoside series (GM3, GD3), and sialosylparagloboside in the molar ratio of 54:40:6, respectively. Testicular GM3, GM1, GD3, GD1a, GD1b and GT1b ran as double bands on thin-layer chromatography which could be accounted for by observed differences in the fatty acid moiety. In addition, the slower migrating band of each pair contained some or all of its sialic acid residues as N-glycolylneuraminic acid, whereas the faster migrating band contained exclusively N-acetylneuraminic acid, except for GM3 where N-acetylneuraminic acid was the sole species in both bands. Thin-layer chromatography of sperm gangliosides revealed seven bands comigrating with equivalent testicular gangliosides. These coincided with the slower migrating bands of testicular GM3, GM1, GD3, GD1a, both bands of GD1b, and possibly both bands of GT1b. Sperm contained only trace amounts of sialosylparagloboside but, in addition, two unidentified bands which were absent from testis were also observed. The molar ratio of the ganglio series to the hematoside series in sperm was 42:58 with GM3 accounting for 42% of total gangliosides.     

Ganglioside modulation regulates epithelial cell adhesion and spreading via ganglioside-specific effects on signaling

Gangliosides are implicated in regulating cell adhesion and migration on fibronectin by binding with the alpha(5) subunit of alpha(5)beta(1) integrin. However, the effects of gangliosides on cell spreading and related signaling pathways are unknown. Increases in gangliosides GT1b and GD3 inhibited spreading on fibronectin, concurrent with inhibition of Src and focal adhesion kinase. Although antibody blockade of GT1b or GD3 function and gene-modulated ganglioside depletion stimulated spreading and activated Src and focal adhesion kinase, the augmented spreading by disruption of GT1b function, but not by disruption of GD3 function, was inhibited by blockade of Src and focal adhesion kinase activation. In contrast, inhibitors of protein kinase C prevented the stimulation of spreading by GD3 functional inhibition, but not by GT1b functional blockade. Modulation of either GT1b or GD3 content affected phosphoinositol 3-kinase activation, and inhibition of this activation reversed the stimulation of cell spreading by anti-GD3 antibody, anti-GT1b antibody, and ganglioside depletion, suggesting that phosphoinositol 3-kinase is an intermediate in both the FAK/Src and protein kinase C pathways that lead to cell spreading. These studies demonstrate that epithelial cell ganglioside GT1b modulates cell spreading through alpha(5)beta(1)/FAK and phosphoinositol 3-kinase signaling, whereas GD3-modulated spreading appears to involve phosphoinositol 3-kinase-dependent protein kinase C signaling.     

Helicobacter pylori encoding the pathogenicity island activates matrix metalloproteinase 1 in gastric epithelial cells via JNK and ERK

Helicobacter pylori colonizes the human gastric epithelium and induces an inflammatory response that is a trigger for gastric carcinogenesis. Matrix metalloproteinases (MMPs) have recently been shown to be up-regulated in gastric epithelial cells infected with H. pylori and might contribute to the pathogenesis of peptic ulcer. The aim of this study was to extend the knowledge about the effect of H. pylori infection on MMP-1 expression by gastric epithelial cells, the kinetics of induction, the pathogenetic properties of the bacterium, and the intracellular signaling pathways required for MMP-1 up-regulation. Expression of MMP-1 was induced more than 10-fold by co-culture of AGS+cells with H. pylori strains carrying the pathogenicity island (PAI). H. pylori strains with mutations in the PAI and a defective type IV secretion system had no effect on MMP-1. Double immunofluorescence revealed strong MMP-1 staining in epithelial cells of gastric biopsies at sites of bacterial attachment. In vitro, MMP-1 is up-regulated by interleukin-1beta and tumor necrosis factor-alpha, but these regulatory mechanisms are not operating in H. pylori infection as shown by inhibitory antibodies. Specific inhibitors of JNK kinase and ERK1/2 kinase were found to suppress the H. pylori-induced MMP-1 expression and activity. AGS cells treated with antisense MMP-1 showed a significantly reduced potential to degrade reconstituted basement membrane. Our results suggest that in gastric epithelial cells, H. pylori up-regulates MMP-1 in a type IV secretion system-dependent manner via JNK and ERK1/2. Induction of MMP-1 is further implicated in complex processes induced by H. pylori, resulting in tissue degradation and remodeling of the gastric mucosa.     

Substrate specificity and inhibitor studies of a membrane-bound ganglioside sialidase isolated from human brain tissue

A ganglioside-specific sialidase that controls cellular functions such as growth, differentiation, and adhesion has been observed in a variety of cells, but its characterization proved difficult due to firm membrane attachment and lability of the purified enzyme. Here we report on the specificity toward gangliosides and susceptibility to certain inhibitors of a ganglioside sialidase solubilized and purified 5100-fold from human brain. The sialidase removed terminal sialic acids from gangliosides GM3, GM4, GD3, GD2, GD1 a, GD1 b, GT1 b and GQ1 b, but was inactive toward gangliosides with sialic acid in a branching position (as in GM1 and GM2). Lyso-GM3 and -GD1a were good substrates, too, whereas O-acetylation of the sialic acid as in 9-O-acetyl-GD3 caused strongly reduced cleavage. The new influenza virus drug 4-guanidino-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Zanamivir) exhibited an IC50 value of about 7 x 10(-5) M that was in the range of the 'classical' sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid; the bacterial sialidase inhibitor 4-nitrophenyloxamic acid, however, was ineffective. The glycosaminoglycans heparan sulfate, heparin, chondroitin sulfates A and B, as well as dextran sulfate and suramin, were all strongly inhibitory, suggesting that glycosaminoglycans present on the cell surface or in the extracellular matrix may influence the ability of the sialidase to alter the ganglioside composition of the membrane.     

A new thin-layer chromatographic approach for separation of multisialogangliosides: Novel gangliosides fractions in the embryonic chicken brain

A novel thin-layer chromatographic procedure has been developed that permits rapid, high-resolution separation of complex ganglioside mixtures and direct densitometric quantification. A special advantage of the new procedure, performed by two different consecutive runs on high-performance thin-layer chromatography plates, is an excellent separation of multisialogangliosides containing more than three sialic acid residues. Using the new procedure, 10 unidentified fractions were detected in embryonic chick brains. These gangliosides were clearly distinguishable from the known gangliosides, GM1, GD3, GD1a, GD2, GD1b, GT1b, and GQ1b. Eight of these “additional” fractions were also found in the brains of rays. From published data on the cod fish brain, 6 of the novel fractions are suggested to correspond to GT3, GT2, GT1c, GQ1c, GP1c, and GP1b. Four fractions, moving on thin-layer chromatography plates below the suggested GP1c have not been reported previously in any vertebrate. Due to their very slow migration rates they may contain gangliosides with six, seven, or more sialic acid residues. During development of the chicken, the relative amounts of the newly detected fractions decrease in favor of GT1b and GD1a.