Sulfated glycoconjugates are powerful modulators of bovine sperm adhesion and release from the oviductal epithelium in vitro

The mechanisms of sperm adhesion and release within the mammalian oviduct are still poorly understood. In this in vitro study, a previously developed adhesion assay was used to analyze the effects of heparin, N-desulfated heparin, fucoidan, dextran sulfate, and dextran on bovine sperm-oviductal cell adhesion and release. Results showed that 1) all sulfated glycoconjugates were powerful inhibitors of sperm binding to oviductal monolayers in a dose-dependent manner, whereas N-desulfated heparin and dextran had no effect; 2) sperm pretreatment with heparin and fucoidan markedly inhibited adhesion; 3) treatment of oviductal monolayers with heparinase I, II, or sodium chlorate (an inhibitor of sulfation) had no effect on sperm adhesion; 4) sulfated glycoconjugates were also powerful and quick inducers of sperm release from oviductal monolayers; and 5) addition of sulfated glycoconjugates to the cocultures caused a sudden increase of bound-sperm flagellar beat frequencies, followed by a release of highly motile sperm. In conclusion, these data support the hypothesis that sulfated glycoconjugates may act as signals that induce sperm release and migration from the oviductal reservoir.     

Inhibition of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> adhesion to Kato III cells by intact and low molecular weight acharan sulfate

We investigated the inhibitory activity of glycosaminoglycans (GAGs) in terms of growth, adhesion, and VacA vacuolation of Helicobacter pylori. Intact acharan sulfate (AS, MW:114 kDa) potently inhibited H. pylori adhesion to Kato III cells with IC₅₀ value of 1.4 mg/mL, while other GAGs did not show any inhibitory activity except for heparin which is a well-known inhibitor of H. pylori adhesion. To investigate whether low molecular weight acharan sulfate (LMWAS) can inhibit H. pylori adhesion, we performed chemical depolymerization of AS by radical reactions to obtain LMWAS. Its physicochemical properties were characterized by high-performance size exclusion chromatography (HPSEC), agarose gel electrophoresis, disaccharide compositional analysis after digestion with heparinase II, and ¹H-NMR spectroscopy. The most potent molecular size of LMWAS was 3 kDa with IC₅₀ value of 32 μg/mL, which is 44-fold more potent than intact AS. These results suggest that AS as well as other GAGs can be chemically depolymerized by free radicals and LMWAS compared to intact AS can be applied as a pharmaceutical candidate in order to inhibit H. pylori adhesion to Kato III cells.     

Effect of sulfated glycoconjugates on capacitation and the acrosome reaction of bovine and hamster spermatozoa

The effects of sulfated glycoconjugates on the preparation of mammalian sperm for fertilization were investigated. The three sulfated glycoconjugates tested were heparin, dextran sulfate, and the fucose sulfate glycoconjugate (FSG) from the sea urchin egg jelly coat. In vivo, FSG induces the acrosome reaction in sea urchin sperm. Bovine sperm were found to be capacitated by heparin and FSG as judged both by ability of lysophosphatidylcholine (LC) to induce an acrosome reaction and by ability to fertilize bovine oocytes in vitro. The mechanism by which heparin or FSG capacitated bovine sperm appeared similar, since glucose inhibited capacitation by both glycoconjugates. In contrast to effects on bovine sperm, heparin and FSG induced the acrosome reaction in capacitated hamster sperm. When hamster sperm were incubated under noncapacitating conditions, heparin had no effect on capacitation or the acrosome reaction. Three molecular weights (MW) of dextran sulfate (5,000, 8,000, 500,000) were found to capacitate bovine sperm as judged by the ability of LC to induce an acrosome reaction. Whereas bovine sperm incubated with 5,000 or 8,000 M W dextran sulfate fertilized more bovine oocytes than control sperm (P <0.05), sperm treated with 500,000 M W dextran sulfate failed to penetrate oocytes. The high-MW dextran sulfate appeared to interact with the zona pellucida and/or sperm to prevent sperm binding. Results suggest that sulfated glycoconjugates may prepare sperm for fertilization across a wide range of species.     

Heparin Promotes Suspension Adaptation Process of CHO–TS28 Cells by Eliminating Cell Aggregation

While heparin has been shown to eliminate cell aggregation in suspension adaptations of insect and HEK293 cells for virus-based cell cultures, the role of heparin in long period serum-free suspension adaptation of the anchorage-dependent Chinese hamster ovary (CHO) cell lines remains inconclusive. In this paper, we explore the potential application of heparin in suspension adaptation of CHO cell line which produces an anti-human chimeric antibody cHAb18. Heparin showed a concentration-dependent inhibition of CHO–TS28 cell-to-cell adhesion, with a significant inhibitory effect occurring when the concentration exceeded 250 μg/ml (P < 0.001). Heparin also exhibited a cell aggregation elimination role at all concentrations (P < 0.001). Furthermore, heparin promoted cell growth and antibody secretion, with the highest cell density ((99.83 ± 12.21) × 10⁴ cells/ml, P = 0.034) and maximum antibody yield ((9.46 ± 0.94) mg/l, P < 0.001) both occurring at 250 μg/ml heparin. When agitated, cell aggregates were effectively dispersed by 250 μg/ml heparin and a single-cell suspension culture process was promoted. In suspension adapted CHO–TS28 cells, cell growth rates and specific antibody productivity were maintained; while antigen-binding activity improved slightly. Together, our results show that heparin may promote suspension adaptation of anchorage-depended CHO cells by resisting cell aggregation without reducing cell growth, antibody secretion, and antigen-binding activity.     

Interactions of a laminin-binding peptide from a 33-kDa protein related to the 67-kDa laminin receptor with laminin and melanoma cells are heparin-dependent.

A laminin-binding peptide (peptide G), predicted from the cDNA sequence for a 33-kDa protein related to the 67-kDa laminin receptor, specifically inhibits binding of laminin to heparin and sulfatide. Since the peptide binds directly to heparin and inhibits interaction of another heparin-binding protein with the same sulfated ligands, this inhibition is due to direct competition for binding to sulfated glycoconjugates rather than an indirect effect of interaction with the binding site on laminin for the 67-kDa receptor. Direct binding of laminin to the peptide is also inhibited by heparin. This interaction may result from contamination of the laminin with heparan sulfate, as binding is enhanced by the addition of substoichiometric amounts of heparin but inhibited by excess heparin and two heparin-binding proteins. Furthermore, laminin binds more avidly to a heparin-binding peptide derived from thrombospondin than to the putative receptor peptide. Adhesion of A2058 melanoma cells on immobilized peptide G is also heparin-dependent, whereas adhesion of the cells on laminin is not. Antibodies to the beta 1-integrin chain or laminin block adhesion of the melanoma cells to laminin but not to peptide G. Thus, the reported inhibition of melanoma cell adhesion to endothelial cells by peptide G may result from inhibition of binding of laminin or other proteins to sulfated glycoconjugate receptors rather than from specific inhibition of laminin binding to the 67-kDa receptor.     

Review: Prospects for the Use of Extracts and Polysaccharides from Marine Algae to Prevent and Treat the Diseases Caused by Helicobacter pylori

Helicobacter pylori possesses a broad spectrum of pathogenic factors that allow it to survive and colonize the gastric mucosa, and thus, the pathogenetic targets, which have the same diversity, require search for and the development of alternative, effective, and innocuous means for the eradication of H. pylori. In recent years, fucoidans have been extensively studied due to the numerous interesting biological activities, including the anti‐adhesive, anti‐oxidative, antitoxic, immunomodulatory, anticoagulant, and anti‐infection effects. This review summarizes the data on the effects of extracts and sulfated polysaccharides of marine algae, mainly fucoidans, on pathogenic targets in Helicobacter infection. The pathogenetic targets for therapeutic agents after H. pylori infection, such as flagellas, urease, and other enzymes, including adhesins, cytotoxin A (VacA), phospholipase, and L‐8, are characterized here. The main target for the sulfated polysaccharides of seaweed is cell receptors of the gastric mucosa. This review presents the published data about the pleiotropic anti‐inflammatory effects of polysaccharides on the gastric mucosa. It is known that fucoidan and other sulfated polysaccharides from algae have anti‐ulcer effects, prevent the adhesion of H. pylori to, and reduce the formation of biofilm. The authors speculate that the effect of sulfated polysaccharides on the infectious process caused by H. pylori is related to their action on innate and adaptive immunity cells, and also anti‐oxidant and antitoxic potential. Presented in the review are materials indicated for the study of extracts and sulfated polysaccharides from seaweed during H. pylori infection, as these compounds are characterized by multimodality actions. Based on the analysis of literary materials in recent years, the authors concluded that fucoidan can be attributed to the generation of new candidates to create drugs intended for the inclusion in the scheme of eradication therapy of H. pylori infection.     

Malaria sporozoites and circumsporozoite proteins bind specifically to sulfated glycoconjugates

Circumsporozoite (CS) proteins, which densely coat malaria (Plasmodia) sporozoites, contain an amino acid sequence that is homologous to segments in other proteins which bind specifically to sulfated glycoconjugates. The presence of this homology suggests that sporozoites and CS proteins may also bind sulfated glycoconjugates. To test this hypothesis, recombinant P. yoelii CS protein was examined for binding to sulfated glycoconjugate-Sepharoses. CS protein bound avidly to heparin-, fucoidan-, and dextran sulfate-Sepharose, but bound comparatively poorly to chondroitin sulfate A- or C-Sepharose. CS protein also bound with significantly lower affinity to a heparan sulfate biosynthesis-deficient mutant cell line compared with the wild-type line, consistent with the possibility that the protein also binds to sulfated glycoconjugates on the surfaces of cells. This possibility is consistent with the observation that CS protein binding to hepatocytes, cells invaded by sporozoites during the primary stage of malaria infection, was inhibited by fucoidan, pentosan polysulfate, and heparin. The effects of sulfated glycoconjugates on sporozoite infectivity were also determined. P. berghei sporozoites bound specifically to sulfatide (galactosyl[3-sulfate]beta 1-1ceramide), but not to comparable levels of cholesterol-3-sulfate, or several examples of neutral glycosphingolipids, gangliosides, or phospholipids. Sporozoite invasion into hepatocytes was inhibited by fucoidan, heparin, and dextran sulfate, paralleling the observed binding of CS protein to the corresponding Sepharose derivatives. These sulfated glycoconjugates blocked invasion by inhibiting an event occurring within 3 h of combining sporozoites and hepatocytes. Sporozoite infectivity in mice was significantly inhibited by dextran sulfate 500,000 and fucoidan. Taken together, these data indicate that CS proteins bind selectively to certain sulfated glycoconjugates, that sporozoite infectivity can be inhibited by such compounds, and that invasion of host hepatocytes by sporozoites may involve interactions with these types of compounds.     

Inhibition of lactoferrin and vitronectin binding toStaphylococcus aureus by heparin

Cells ofStaphylococcus aureus strain ISP 546, previously shown to express high binding of vitronectin (Vn), were shown also to bind radioactive iodine-labeled human lactoferrin (Lf). The binding was specific, and heat treatment ofS. aureus cells abolished Lf binding. When cells were preincubated with heparin (2.5 g/ml), binding of Vn and Lf was inhibited by more than 90%. Two other sulfated carbohydrate polymers, fucoidan and dextran sulfate (mol weights 5000 and 8000) inhibited binding of both iodine-labeled Vn and Lf by about 75% each. No synergistic inhibitory effect between heparin and fuocidan on Lf binding byS. aureus was detected. Mannose, mannoseamine and poly--sialic acid (colominic acid) inhibited Vn binding by about 40% but had no effect on Lf binding. A cell surface extract of ISP 546 inhibited Vn binding but did not affect Lf binding. Lf binding toS. aureus was inhibited by preincubation of the cells with Vn, and Vn binding toS. aureus was inhibited by preincubation with Lf. Our data indicate that Vn and Lf bind to different molecules on theS. aureus cell surface, involving heparin and other carbohydrate interactions.     

Heparin-induced aggregation of lymphoid cells

The effects of different carbohydrates on cell-to-cell adhesion were examined in an aggregation assay, which consisted of swirling a suspension of cells and monitoring the loss of single cells with a Coulter Counter. Of the carbohydrates tested, only heparin and dextran sulfate induced cell aggregation. This effect occurred in freshly isolated mouse splenocytes and in cultured cells of lymphoid origin (P388, YAA-CI) but not in cell lines of fibroblastic origins (3T3, SV-3T3, BHK, and PY-BHK). Using the YAA-CI cell line for further study, we found that aggregation could be induced by relatively small amounts of heparin (less than 10 micrograms/ml). Binding experiments with 3H-heparin showed that under normal physiological conditions each YAA-CI cell bound approximately 2 X 10(6) molecules of heparin at saturation with a Kd of 3.5 X 10(-7) M. This binding was blocked by both unlabelled heparin and dextran sulfate but not by other carbohydrates. When the pH of the medium was decreased, the heparin-induced aggregation was inhibited, and the Kd of the 3H-heparin binding was increased. In a similar fashion, when the ionic strength of the medium was increased, heparin-induced aggregation was inhibited and the Kd of the interaction was increased. These results suggest that the aggregation is inversely related to the Kd of the interaction and that the binding of heparin to the cell surface is primarily of an ionic nature.     

A role of mast cell glycosaminoglycans for the immunological expulsion of intestinal nematode, Strongyloides venezuelensis

We examined effects of mast cell glycosaminoglycans on the establishment of the intestinal nematode, Strongyloides venezuelensis, in the mouse small intestine. When intestinal mastocytosis occurred, surgically implanted adult worms could not invade and establish in the intestinal mucosa. In mast cell-deficient W/Wv mice, inhibition of adult worm invasion was not evident as compared with littermate +/+ control mice. Mucosal mastocytosis and inhibition of S. venezuelensis adult worm mucosal invasion was tightly correlated. To determine effector molecules for the invasion inhibition, adult worms were implanted with various sulfated carbohydrates including mast cell glycosaminoglycans. Among sulfated carbohydrates tested, chondroitin sulfate (ChS)-A, ChS-E, heparin, and dextran sulfate inhibited invasion of adult worms into intestinal mucosa in vivo. No significant inhibition was observed with ChS-C, desulfated chondroitin, and dextran. ChS-E, heparin, and dextran sulfate inhibited adhesion of S. venezuelensis adult worms to plastic surfaces in vitro. Furthermore, binding of intestinal epithelial cells to adhesion substances of S. venezuelensis, which have been implicated in mucosal invasion, was inhibited by ChS-E, heparin, and dextran sulfate. Because adult worms of S. venezuelensis were actively moving in the intestinal mucosa, probably exiting and reentering during infection, the possible expulsion mechanism for S. venezuelensis is inhibition by mast cell glycosaminoglycans of attachment and subsequent invasion of adult worms into intestinal epithelium.     

Binding of Selected Extracellular Matrix Proteins to Enterococci and Streptococcus bovis of Animal Origin

Thirty-three enterococcal strains and 10 Streptococcus bovis strains were investigated for their protein-binding cell surface components. Seven extracellular matrix (ECM) proteins were immobilized on Difco latex beads to detect these components on the surface of all enterococcal strains and eight non-autoaggregating S. bovis strains by a particle agglutination assay (PAA). Twenty-three selected strains were also examined in microtiter plate assays. According to the absorbance readings (A_570nm), 11 strains were classified as nonadherent (A_570nm < 0.1), 10 strains as weakly adherent (0.1 < A_570nm > 0.3), and 2 strains as strongly adherent (A_570nm > 0.3) in these assays. A direct correlation was found between the values obtained in PAA and A_570nm readings of microtiter plate assays. Binding of ¹²⁵I-labeled bovine lactoferrin to enterococci and streptococci was in the range of 6%–30% and of ¹²⁵I-labeled human vitronectin in the range of 9%–33% to streptococci. The binding of ¹²⁵I-labeled ECM proteins to selected strains was much more effectively inhibited by sulfated carbohydrates than by non-sulfated hyaluronic acid, indicating the importance of the sulfate groups of these inhibitors. An inhibition effect of heparin on bLf binding to four selected strains was higher in comparison with fucoidan in the microtiter plates. Thirty-five out of 44 strains had agglutinated rabbit erythrocytes. However, these strains showed no ability to agglutinate bovine or sheep erythrocytes. Received: 28 April 1999 / Accepted: 26 July 1999     

Sulfated Polysaccharides Are Required for Collagen-Induced Vascular Tube Formation

We have previously shown that soluble type I collagen can induce vascular tube formation when it contacts the apical side of a confluent endothelial monolayer. In this study we have examined which soluble agent(s) are required for collagen-induced tube formation. Human neo-natal foreskin microvascular endothelial cells, maintained in basal medium, were preincubated with each test agent for 2 h prior to the addition of solubilised type I collagen (100 μg/ml). After 6 h, tube formation was quantitated using image analysis and expressed as the mean area of tube formation (mm²) per microscopic field of view. Collagen-induced tube formation did not occur in the presence of endothelial cell growth supplement, basic fibroblast growth factor, or normal pooled human serum. In contrast, the addition of heparin at 5 or 50 μg/ml caused extensive tube formation (0.22 ± 0.07 and 0.30 ± 0.12 mm², respectively) whereas at 500 μg/ml little tube formation occurred (0.03 ± 0.02 mm²). Protamine sulfate, an antagonist of heparin, inhibited collagen-induced tube formation in a dose-dependent manner. Pentosan polysulfate, dextran sulfate, heparan sulfate, and chondroitin sulfate mimicked the action of heparin. Partially sulfated heparin (de-N-sulfated heparin) stimulated less tube formation compared to heparin (0.15 ± 0.06 mm² at 50 μg/ml). The nonsulfated polysaccharides, xylan and dextran, had no effect on tube formation. In summary, sulfated polysaccharides are required for collagen-induced vascular tube formation in vitro. The sulfation of these molecules appears to be vital for collagen-induced tube formation.     

Beneficial effect of Burdock complex on asymptomatic Helicobacter pylori‐infected subjects: A randomized,double‐blind placebo‐controlled clinical trial

Background

Burdock complex (BC) constitutes of burdock (Arctium lappa), angelica (Angelica sinensis), gromwell (Lithospermum erythrorhizon), and sesame (Sesamum indicum) oil, which are commonly used in traditional Chinese medicine (TCM) for treating various disorders. This study intended to examine the anti‐H. pylori activity of BC on AGS cell model as well as in asymptomatic H. pylori‐infected subjects.

Materials and Methods

AGS cell incubated with H. pylori and treated with BC to evaluate the minimum inhibition concentration (MIC), cell viability (MTT) anti‐adhesion activity, and inflammatory markers. In case of clinical trial, H. pylori‐positive subjects (urea breath test [UBT] >10%, n = 36) were enrolled and requested to intake BC (n = 19) or placebo (n = 17) for 8 weeks. Antioxidant capacity, total phenol, UBT, inflammatory markers were analyzed at the initial, 4th, 8th, and 10th weeks. Moreover, the endoscopic examination was carried out on baseline and 10th week.

Results

In vitro studies showed that BC treatment significantly inhibited (P < .05) the inflammatory markers and adhesion of H. pylori to AGS cell. However, H. pylori‐infected subject ingested with BC for 8 weeks significantly decreased (P < .05) the UBT value, inflammatory markers with improved antioxidant activity, and phenolic levels as compared to placebo. Also, consumption of BC considerably healed the ulcer wound.

Conclusion

Overall, the BC could attenuate H. pylori infection by inhibiting H. pylori adhesion and subsequent inflammatory response on the gastric epithelial cell (AGS) as well as clinically ameliorated UBT, antioxidant capacity, and alleviated inflammation to display its anti‐H. pylori activity.     

Helicobacter pylori Induces Apoptosis of T- and B-Cell Lines and Translocates Mitochondrial Apoptosis-Inducing Factor to Nucleus

Immune cell apoptosis may play a role in human persistent Helicobacter pylori infection. We planned to study the apoptosis of T and B cells by H. pylori strains. T (Jurkat) and B (Raji) cell lines were co-cultured with cagA-positive H. pylori strains carrying different vacA genotypes (s1a/m1, s1a/m2, and s2/m2). Apoptosis was detected by microscopy, DNA fragmentation assay, and flow cytometry. Apoptosis-inducing factor (AIF) transfer from mitochondria to nucleus was studied by immunoblot analysis. Apoptosis of T and B cells was significantly higher in H. pylori-infected cells than in uninfected controls (s1a/m1 80%, s1a/m2 78%, s2m2 69% vs. control 16% for T cells, P < 0.001; s1 a/m1 78%, s1a/m2 73%, s2m2 62% vs. control 24% for B cells, P < 0.001 by flow cytometry) with no difference among the genotypes. AIF transfer from mitochondria to nucleus was demonstrated in both apoptotic cell lines. Thus, H. pylori induces apoptosis in T- and B-cell lines and translocates AIF. T and B cells deletion through apoptosis may explain the persistence of H. pylori infection; its role in pathogenesis needs further research.     

Helicobacter pylori in Liquid Culture: Evaluation of Growth Rates and Ultrastructure

This study investigated the growth of Helicobacter (H.) pylori in Brucella broth supplemented with either IsoVitaleX (1% vol/vol), hemin (0.1% wt/vol), agar (0.3% wt/vol), or blood agar blocks (1.5% wt/vol agar). IsoVitaleX was found to significantly shorten the lag phase, while hemin inhibited the growth within the first 24 hours but later acted as a growth stimulant. There was a tendency toward stronger growth when blood agar blocks were added to the medium. Subsequent electron microscopic evaluation revealed that cells of H. pylori were attached to blood agar block surfaces. In contrast, the supplementation of Brucella broth with agar did not significantly increase the cell density. When H. pylori was grown in the presence of IsoVitaleX, strongly stainable electron-dense bodies (140–200 nm) were seen in the cytoplasms. Incubation of cultures on rotary shakers at 120 rpm significantly enhanced growth. The addition of glycerol (15% vol/vol) or fetal bovine serum (15% vol/vol) showed good ultrastructural preservation of bacteria with undamaged cell walls and cytoplasmic membranes, and the cytoplasms were ribosome-dense. Cell counts revealed that cultures stored in glycerol or fetal bovine serum had a significantly lower loss in viability when compared with cultures stored without cryopreservatives. Unprotected cells of H. pylori showed on electron micrographs clumping, cell lysis, and flagellar damage. Finally, the survival rates of H. pylori after multiple thawing from storage at −80°C were best in Brucella broth/glycerol, Brucella broth/fetal bovine serum, and Brucella broth without cryopreservative (in descending order). Received: 10 November 1997 / Accepted: 29 January 1998     

EPs® 7630, an extract from Pelargonium sidoides roots inhibits adherence of Helicobacter pylori to gastric epithelial cells

Helicobacter pylori specifically adheres to gastric host cells, mainly based on carbohydrate-mediated cell–cell interaction. The extract of Pelargonium sidoides roots (EPs^® 7630), a South African herbal remedy, is currently used to treat acute bronchitis. EPs^® 7630 prevents bacteria from attaching to cell membranes. Therefore, the ability of EPs^® 7630 to interfere with H. pylori growth and adhesion to gastric epithelial cells (AGS cells) was tested in vitro. EPs^® 7630 inhibited H. pylori growth and with higher potency adhesion to gastric AGS cells. EPs^® 7630 (50 and 100 μg/ml) reduced bacterial count attached to AGS cells by 77% and 91%, respectively. The results suggest that the mode of action of EPs^® 7630 is mainly related to its anti-adhesive activity.     

Effects of Extremely Low-Frequency Electromagnetic Fields on Helicobacter pylori Biofilm

The aim of this work was to investigate the effects of exposure to extremely low-frequency electromagnetic fields (ELF-EMF) both on biofilm formation and on mature biofilm of Helicobacter pylori. Bacterial cultures and 2-day-old biofilm of H. pylori ATCC 43629 were exposed to ELF-EMF (50 Hz frequency–1 mT intensity) for 2 days to assess their effect on the cell adhesion and on the mature biofilm detachment, respectively. All the exposed cultures and the respective sham exposed controls were studied for: the cell viability status, the cell morphological analysis, the biofilm mass measurement, the genotypic profile, and the luxS and amiA gene expression. The ELF-EMF acted on the bacterial population during the biofilm formation displaying significant differences in cell viability, as well as, in morphotypes measured by the prevalence of spiral forms (58.41%) in respect to the controls (33.14%), whereas, on mature biofilm, no significant differences were found when compared to the controls. The measurement of biofilm cell mass was significantly reduced in exposed cultures in both examined experimental conditions. No changes in DNA patterns were recorded, whereas a modulation in amiA gene expression was detected. An exposure to ELF-EMF of H. pylori biofilm induces phenotypic changes on adhering bacteria and decreases the cell adhesion unbalancing the bacterial population therefore reducing the H. pylori capability to protect itself.     

Role of Cell Surface O-Linked Oligosaccharides in Adhesion of HL60 Cells to Fibronectin: Regulation of Integrin-Dependent Cell Adhesion by O-Linked Oligosaccharide Elongation

The adhesion of the myelogenous leukemia cell line, HL60, to fibronectin and its fragments, heparin binding fragment (40 kDa) and cell attachment fragment (120 kDa), was enhanced by culturing with benzyl-α-GalNAc (BZαGalNAc). Enhancement of cell adhesion to fibronectin was also observed on treatment of HL60 cells with 12-O-tetradecanoylphorbol 13-acetate (TPA). However, an additive effect of BZαGalNAc and TPA treatments was not observed. The expression of VLA4 and VLA5 did not change during treatment with BZαGalNAc or TPA. Cell adhesion to fibronectin before and after treatment with BZαGalNAc or TPA was inhibited by anti-VLA4 and anti-VLA5 monoclonal antibodies. Staining of the cells with Helix pomatia lectin demonstrated that culturing of the cells with BZαGalNAc blocked elongation of O-linked oligosaccharides on the cell surface and led to accumulation of GalNAc-O-Ser/Thr. Labeling of cell surface carbohydrates with [³H]-glucosamine followed by treatment with TPA revealed that O-glycosylated glycoproteins including CD43 were released from the cell surface during this treatment. These findings indicate that integrin-dependent cell adhesion, particularly VLA4- or VLA5-dependent cell adhesion, of HL60 cells is prevented with the extension of O-linked oligosaccharides and recovers with the disappearance of O-linked oligosaccharides from the cell surface.     

<Emphasis Type="Italic">H. pylori</Emphasis> related proinflammatory cytokines contribute to the induction of miR-146a in human gastric epithelial cells

MicroRNAs have been implicated as a central regulator of the immune system. We have previously reported that Helicobacter pylori (H. pylori) was able to increase the expression of miR-146a, and miR-146a may negatively regulate H. pylori-induced inflammation, but the exact mechanism of how H. pylori contribute the induction of miR-146a is not clear. Here, we attempted to assess the role of H. pylori related proinflammatory cytokines including interleukin (IL)-8, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β, and cytotoxin-associated gene A (CagA) virulence factor on the induction of miR-146a. We found that IL-8, TNF-α, and IL-1β could contribute to the induction of miR-146a in gastric epithelial cell HGC-27 in NF-κB-dependent manner, while the induction of miR-146a upon H. pylori stimulation was independent of above proinflammatory cytokines. Furthermore, overexpression of miR-146a reduced H. pylori—induced IL-8, TNF-α, and IL-1β. However, CagA had no effect on the miR-146a induction. Taken together, our study suggest that proinflammatory cytokines IL-8, TNF-α, and IL-1β could contribute to the induction of miR-146a during H. pylori infection, while CagA is not necessarily required for miR-146a induction. miR-146a may function as novel negative regulators to modulate the inflammation.