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ABSTRACT: Bacteroides fragilis is found among the normal intestinal flora and is involved in host immunostimulation via TLR2. Its cell surface components, such as LPS and capsular polysaccharides, were reported to participate in host immunostimulation. In this study, we report on the existence of a lipoprotein that acts as a TLR2 stimulant in B. fragilis. The TLR2-stimulating lipoprotein was obtained using Triton X-114-water phase partitioning followed by preparative SDS-PAGE. Its N-terminal hydrophobic peptide, which was separated from a tryptic digest, was characterized as a triacylated lipopeptide, and the lipoprotein was identified as BF1333 by mass spectrometry of Asp-N-digested peptides. These results showed that the lipoprotein acts as a TLR2-stimulating component in B. fragilis.
Innate Immunity 08/2012; · 4.00 Impact Factor
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ABSTRACT: Helicobacter pylori, a Gram-negative bacterium, causes gastroduodenal inflammatory diseases such as chronic gastritis and peptic ulcers, and is also a risk factor for gastric carcinogenesis. In this article, we review recent developments and findings in the chemical synthesis and immunomodulatory activities of H. pylori lipid A and 3-deoxy-D-manno-2-octulosonic acid (Kdo)-lipid A, to clarify the structural basis for the inflammatory response to H. pylori LPS. The synthetic methods include a new divergent synthetic approach with a widely applicable key intermediate for other types of lipid A structures, as well as a selective α-glycosylation reaction between Kdo and lipid A. Cytokine induction assays of the chemically synthesized lipid A structures showed selective cytokine induction depending on the patterns of acyl groups and phosphate groups. The results of cytokine induction assay suggested that H. pylori LPS can modulate the immune response during infection, and also plays a role in chronic inflammatory responses.
Carbohydrate research 03/2012; 356:37-43. · 2.03 Impact Factor
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ABSTRACT: There is increasing evidence that soluble glycosaminoglycans such as heparin can interfere with the infectivity of various viruses, including ecotropic murine leukemia viruses (MLVs). The ecotropic MLV, Friend MLV (F-MLV) and the neuropathogenic variants A8 MLV and PVC-211 MLV, were susceptible to heparin-mediated inhibition of infection of NIH 3T3 cells. To investigate the interaction between the envelope glycoprotein (Env) of MLV and heparin, we prepared vesicular stomatitis virus-based pseudotyped viruses carrying the Env of F-, A8, or PVC-211 MLVs. Surface plasmon resonance analyses indicated that the Env of A8 and PVC-211 MLVs had a higher binding activity to heparin than that of F-MLV. We examined the influence of N- or O-sulfation of heparin on binding activity to Env and on the inhibition of the infectivity of MLV and pseudotyped viruses carrying Env. This analysis indicated that the O-sulfate groups of heparin play a major role in determining Env-dependent inhibitory effects.
Virology 03/2012; 424(1):56-66. · 3.35 Impact Factor
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ABSTRACT: The structural characterization of glycolipids from Thermus thermophilus HB8 was performed in this study. Two neutral and one acidic glycolipids were extracted and purified by the modified TLC-blotting method, after which their chemical structures were determined by chemical composition analysis, mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The structure of one of the neutral glycolipids, NGL-A, was Galp(α1-6)GlcpNacyl(β1-2)Glcp(α1-)acyl(2)Gro, and the other, NGL-C, was Galf(β1-2)Galp(α1-6)GlcpNacyl(β1-2)Glcp(α1-)acyl(2)Gro. The structure of NGL-C was identical to that reported previously [Oshima, M. and Ariga, T. (1976) FEBS Lett. 64, 440]. Both neutral glycolipids shared a common structural unit found in the Thermus species. The acyl groups found in NGL-A and NGL-C, iso-type pentadecanoxy and heptadecanoxy fatty acid, were also the same as those found in this species. In contrast, the acidic glycolipid, AGL-B, possessed the structure of N-(((GlcpNAc(α1-)acyl(2)Gro)P-2)GroA)alkylamine. The alkyl group in AGL-B was an iso-type heptadecanyl, suggesting that the iso-type structure of the long alkyl chain is responsible for the thermal stability of the bacteria.
PLoS ONE 01/2012; 7(7):e35067. · 4.09 Impact Factor
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ABSTRACT: The glycosaminoglycan heparin has been shown to bind to platelet integrin αIIbβ3 and induce platelet activation and aggregation, although the relationship between binding and activation is unclear. We analyzed the interaction of heparin and αIIbβ3 in detail, to obtain a better understanding of the mechanism by which heparin acts on platelets.
We assessed conformational changes in αIIbβ3 by flow cytometry of platelets exposed to unfractionated heparin. In human platelets and K562 cells engineered to express αIIbβ3, we assayed the effect of heparin on key steps in integrin signaling: phosphorylation of the β3 chain cytoplasmic tail, and activation of src kinase. We measured the heparin binding affinity of purified αIIbβ3, and of recombinant fragments of αIIb and β3, by surface plasmon resonance.
Heparin binding results in conformational changes in αIIbβ3, similar to those observed upon ligand binding. Heparin binding alone is not sufficient to induce tyrosine phosphorylation of the integrin β3 cytoplasmic domain, but the presence of heparin increased both β3 phosphorylation and src kinase activation in response to ligand binding. Specific recombinant fragments derived from αIIb bound heparin, while recombinant β3 did not bind. This pattern of heparin binding, compared to the crystal structure of αIIbβ3, suggests that heparin-binding sites are located in clusters of basic amino acids in the headpiece and/or leg domains of αIIb. Binding of heparin to these clusters may stabilize the transition of αIIbβ3 to an open conformation with enhanced affinity for ligand, facilitating outside-in signaling and platelet activation.
Thrombosis Research 12/2011; 129(6):743-9. · 2.44 Impact Factor
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ABSTRACT: Helicobacter pylori is a common cause of gastroduodenal inflammatory diseases such as chronic gastritis and peptic ulcers and also an important factor in gastric carcinogenesis. Recent reports have demonstrated that bacterial inflammatory processes, such as stimulation with H. pylori lipopolysaccharide (LPS), initiate atherosclerosis. To establish the structures responsible for the inflammatory response of H. pylori LPS, we synthesized various kinds of lipid A structures (i.e., triacylated lipid A and Kdo-lipid A compounds), with or without the ethanolamine group at the 1-phosphate moiety, by a new divergent synthetic route. Stereoselective α-glycosylation of Kdo N-phenyltrifluoroacetimidate was achieved by use of microfluidic methods. None of the lipid A and Kdo-lipid A compounds were a strong inducer of IL-1β, IL-6, or IL-8, suggesting that H. pylori LPS is unable to induce acute inflammation. In fact, the lipid A and Kdo-lipid A compounds showed antagonistic activity against cytokine induction by E. coli LPS, except for the lipid A compound with the ethanolamine group, which showed very weak agonistic activity. On the other hand, these H. pylori LPS partial structures showed potent IL-18- and IL-12-inducing activities. IL-18 has been shown to correlate with chronic inflammation, so H. pylori LPS might be implicated in the chronic inflammatory responses induced by H. pylori. These results also indicated that H. pylori LPS can modulate the immune response: NF-κB activation through hTLR4/MD-2 was suppressed, whereas production of IL-18 and IL-12 was promoted.
Chemistry 11/2011; 17(51):14464-74. · 5.93 Impact Factor
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ABSTRACT: Extensin is a glycoprotein that is rich in hydroxyprolines linked to β-L-arabinofuranosides. In this study, we cloned a hypBA2 gene that encodes a novel β-L-arabinobiosidase from Bifidobacterium longum JCM 1217. This enzyme does not have any sequence similarity with other glycoside hydrolase families but has 38-98% identity to hypothetical proteins in Bifidobacterium and Xanthomonas strains. The recombinant enzyme liberated L-arabinofuranose (Araf)-β1,2-Araf disaccharide from carrot extensin, potato lectin, and Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara(3)-Hyp) but not Araf-α1,3-Araf-β1,2-Araf-β1,2-Araf-β-Hyp (Ara(4)-Hyp) or Araf-β1,2-Araf-β-Hyp (Ara(2)-Hyp), which indicated that it was specific for unmodified Ara(3)-Hyp substrate. The enzyme also transglycosylated 1-alkanols with retention of the anomeric configuration. This is the first report of an enzyme that hydrolyzes Hyp-linked β-L-arabinofuranosides, which defines a new family of glycoside hydrolases, glycoside hydrolase family 121.
Journal of Biological Chemistry 02/2011; 286(7):5143-50. · 4.77 Impact Factor
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Errol Wijelath,
Mayumi Namekata,
Jacqueline Murray,
Mai Furuyashiki,
Siyuan Zhang,
Daniel Coan,
Masahiro Wakao,
Robert B Harris, Yasuo Suda,
Lianchun Wang,
Michael Sobel
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ABSTRACT: Heparin and heparin-like molecules are known to modulate the cellular responses to vascular endothelial growth factor-A (VEGF-A). In this study, we investigated the likely mechanisms for heparin's influence on the biological activity of VEGF-A. Previous studies have shown that exogenous heparin's effects on the biological activity of VEGF-A are many and varied, in part due to the endogenous cell-surface heparan sulfates. To circumvent this problem, we used mutant endothelial cells lacking cell-surface heparan sulfates. We showed that VEGF-induced cellular responses are dependent in part on the presence of the heparan sulfates, and that exogenous heparin significantly augments VEGF's cellular effects especially when endogenous heparan sulfates are absent. Exogenous heparin was also found to play a cross-bridging role between VEGF-A(165) and putative heparin-binding sites within its cognate receptor, VEGFR2 when they were examined in isolation. The cross-bridging appears to be more dependent on molecular weight than on a specific heparin structure. This was confirmed by surface plasmon resonance binding studies using sugar chips immobilized with defined oligosaccharide structures, which showed that VEGF-A(165) binds to a relatively broad range of sulfated glycosaminoglycan structures. Finally, studies of the far-UV circular dichroism spectra of VEGF-A(165) showed that heparin can also modulate the conformation and secondary structure of the protein.
Journal of Cellular Biochemistry 10/2010; 111(2):461-8. · 2.87 Impact Factor
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ABSTRACT: The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Neutralizing recombinant human antibodies would provide important agents for immunotherapy on human H5N1 virus infection and definition of the critical mimotope for vaccine development. In this study, we have characterized an anti-H5-specific scFv clone, 3D1 from the human-scFv-displaying phage library. 3D1 blocked the binding of H5-Fc to MDCK cells in flow cytometry and neutralized H5N1 subtype influenza A viruses in a microneutralization assay. Employing a peptide-displaying phage library, Ph.D-12, the mimotope was determined to be at #128-131 and #204-211 of H5, which are silic acid-binding regions. In consistency with this result, 3D1 binds the recombinant sugar-binding domain (#50G-#272E) produced by a baculovirus vector. The 3D1 antibody employs the germline gene VH1-23. As this antibody is the first human anti-H5 scFv clearly defined on the sugar-binding epitope, it allows us to investigate the influence of amino acid substitutions in this region on the determination of the binding specificity to either sialic acid α2,6-galactose (SA α2,6Gal) or sialic acid α2,3-galactose (SA α2,3Gal) providing new insight for the development of effective H5N1 pandemic vaccines.
Journal of biochemistry 10/2010; 148(4):507-15. · 1.95 Impact Factor
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ABSTRACT: Many techniques have been tested for their ability to restore cartilage defects, but several problems still remain in the complete healing of injured cartilage. In our previous study, we found that a carboxymethyl-chitin/beta-tricalcium phosphate (CM-chitin/beta-TCP) composite induced cartilage regeneration in the osteochondral defects of rabbits in vivo. We also found that CM-chitin stimulated peritoneal exudate cells (PEC) in mice and induced several kinds of inflammatory cytokines and transforming growth factor beta-1 (TGF-beta1). In this study, we examined whether CM-chitin is responsible for the induction of chondrogenesis via the production of TGF-beta1 in vitro. The murine pluripotent cell line C3H10T1/2 was maintained as a micromass culture in conditioned medium prepared from PEC stimulated with and without CM-chitin. CM-chitin-conditioned medium induced RNA expression of the chondrogenic-factor Sox9 and the matrix proteins aggrecan, Col2a1, and Comp. Their expression levels were decreased in the presence of anti-TGF-beta1 antibody. The micromass tissues cultured in CM-chitin conditioned medium at day 21 were clearly stained by Toluidine blue or Alcian blue (histological staining) and collagen II antibody (immunohistological staining), showing the expression of acidic glycosaminoglycan and type II collagen. Similar results were observed in micromass tissue stimulated with TGF-beta1 as a positive control. However, no chondrogenesis occurred when CM-chitin was added directly to a C3H10T1/2 cell culture. These results indicated that CM-chitin is a potent inducer of chondrogenesis via the induction of TGF-beta1 in immune cells.
Journal of Biomedical Materials Research Part A 09/2010; 94(4):1034-41. · 2.63 Impact Factor
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ABSTRACT: The monosaccharide moieties found in heparin (HP) and heparan sulfate (HS), glucosamine and two kinds of uronic acids, glucuronic and iduronic acids, were efficiently synthesized by use of glucosamine hydrochloride and glucurono-6,3-lactone as starting compounds. In the synthesis of the disaccharide building block, the key issues of preparation of uronic acids (glucuronic acid and iduronic acid moieties) were achieved in 12 steps and 15 steps, respectively, without cumbersome C-6 oxidation. The resulting monosaccharide moieties were utilized to the syntheses of HP/HS disaccharide building blocks possessing glucosamine-glucuronic acid (GlcN-GlcA) or iduronic acid (GlcN-IdoA) sequences. The disaccharide building blocks were also suitable for further modification such as glycosylation, selective deprotection, and sulfation.
Tetrahedron 05/2010; 66(22):3951-3962. · 3.03 Impact Factor
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ChemBioChem 09/2009; 10(14):2311-5. · 3.94 Impact Factor
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ABSTRACT: To analyse the binding of sugar chains to proteins, viruses and cells, the surface plasmon resonance (SPR) technique is very convenient and effective because it is a real-time, non-destructive detection system. Key to this method is linker compounds for immobilization of the sugar chains to the gold-coated chip for SPR. Also, well-designed fluorescent labelling reagents are essential when analysing the structure of trace amounts of sugar chains derived from natural sources, such as glycoproteins on the surface of specific cells. In this report, we developed a novel linker molecule, named 'f-mono', which has both of these properties: simple immobilization chemistry and a fluorescent label. Since the molecule contains a 2,5-diaminopyridyl group and a thioctic acid group, conjugation with sugar chains can be achieved using the well-established reductive amination reaction. This conjugate of sugar chain and fluorescent linker (fluorescent ligand-conjugate, FLC) has fluorescent properties (ex. 335 nm, em. 380 nm), and as little as 1 microg of FLC can be easily purified using HPLC with a fluorescent detector. MS and MS/MS analysis of the FLC is also possible. As a +2 Da larger MS peak ([M + H + 2](+) ion) was always associated with the theoretical MS peak ([M + H](+)) (due to the reduction of the thioctic acid moiety), the MS peaks of the FLC were easily found, even using unfractionated crude samples. Immobilization of the FLC onto gold-coated chips, and their subsequent SPR analyses were successively accomplished, as had been performed previously using non-fluorescent ligand conjugates.
Journal of biochemistry 04/2009; 146(1):33-41. · 1.95 Impact Factor
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ABSTRACT: By a biopanning method using cell sorter, we quickly isolated an antibody phage clone (S1T-A3) specific to human T-lymphotropic virus type 1-carrying T-cell line S1T from a human single chain Fv (scFv) antibody phage library. This scFv antibody bound to HTLV-1-carrying T-cell lines including MT-2, MT-4 and M8166 other than S1T, but not to non-HTLV-1-carrying T-cell lymphomas such as Jurkat and MOLT4 cells. Interestingly, this antibody induced the cell death on S1T cells very quickly (< 30 min). We tried to identify the target molecules by western blotting and mass spectrometric analysis, revealing that the target antigen was HLA class II DR. The cell death was induced only in dimmer form of scFv (diabody) and at 15-fold lower concentration than that of a fusion protein of scFv and human IgG Fc [(scFv)(2)-Fc] or anti HLA-DR mouse whole antibody L243. Thus, S1T-A3 diabody is a small antibody fragment with agonistic activity to induce cell death through HLA-DR. This is the first report elucidating that diabody specific to HLA-DR is effective to induce the cell death in T-cell malignancy especially adult T-cell leukaemic cell line.
Journal of biochemistry 04/2009; 145(6):799-810. · 1.95 Impact Factor
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ABSTRACT: Staphylococcus aureus is known to activate mammalian immune cells through Toll-like receptor 2 (TLR2). We recently demonstrated that a lipoprotein fraction obtained from S. aureus by Triton X-114 phase partitioning is a potent activator of TLR2. In this study, we separated TLR2-activating lipoproteins expressed in S. aureus and characterized an N-terminal structure. The lipoprotein fraction of S. aureus was prepared by glass bead disruption followed by Triton X-114 phase partitioning. The TLR2-activating molecules were mainly detected in the mass range of 30-35 kDa. Seven lipoproteins were identified by the mass spectra of their tryptic digests. Among them, three lipoproteins were separated by preparative SDS-PAGE and proved to activate TLR2. After digestion with trypsin in the presence of sodium deoxycholate, the N terminus of the lipopeptide was isolated from lipoprotein SAOUHSC_02699 by normal phase high pressure liquid chromatography and characterized as an S-(diacyloxypropyl)cystein-containing peptide using tandem mass spectra. The synthetic lipopeptide counterpart also stimulated the cells via TLR2. These results showed that the diacylated lipoprotein from S. aureus acts as a TLR2 ligand in mammalian cells.
Journal of Biological Chemistry 03/2009; 284(14):9147-52. · 4.77 Impact Factor
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ABSTRACT: Recently, we have identified two 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transporters (PAPST1 and PAPST2), which contribute to PAPS transport into the Golgi, in both human and Drosophila. Mutation and RNA interference (RNAi) of the Drosophila PAPST have shown the importance of PAPST-dependent sulfation of carbohydrates and proteins during development. However, the functional roles of PAPST in mammals are largely unknown. Here, we investigated whether PAPST-dependent sulfation is involved in regulating signaling pathways required for the maintenance of mouse embryonic stem cells (mESCs), differentiation into the three germ layers, and neurogenesis. By using a yeast expression system, mouse PAPST1 and PAPST2 proteins were shown to have PAPS transport activity with an apparent K(m) value of 1.54 microM or 1.49 microM, respectively. RNAi-mediated knockdown of each PAPST induced the reduction of chondroitin sulfate (CS) chain sulfation as well as heparan sulfate (HS) chain sulfation, and inhibited mESC self-renewal due to defects in several signaling pathways. However, we suggest that these effects were due to reduced HS, not CS, chain sulfation, because knockdown of mouse N-deacetylase/N-sulfotransferase, which catalyzes the first step of HS sulfation, in mESCs gave similar results to those observed in PAPST-knockdown mESCs, but depletion of CS chains did not. On the other hand, during embryoid body formation, PAPST-knockdown mESCs exhibited abnormal differentiation, in particular neurogenesis was promoted, presumably due to the observed defects in BMP, FGF and Wnt signaling. The latter were reduced as a result of the reduction in both HS and CS chain sulfation. We propose that PAPST-dependent sulfation of HS or CS chains, which is regulated developmentally, regulates the extrinsic signaling required for the maintenance and normal differentiation of mESCs.
PLoS ONE 01/2009; 4(12):e8262. · 4.09 Impact Factor
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ABSTRACT: To obtain lectins without tedious purification steps, we developed a convenient method for a one-step purification of lectins using sugar-immobilized gold nano-particles (SGNPs). Proteins in crude extracts from plant materials were precipitated with 60% ammonium sulphate, and the precipitate was re-dissolved in a small volume of phosphate buffer. The resultant solution was then mixed with appropriate SGNPs under an optimized condition. After incubating overnight at 4 degrees C, lectins in the mixture formed aggregate with SGNPs, which was visually detected and easily sedimented by centrifugation. The aggregate was dissolved by adding inhibitory sugars, which were identical to the non-reducing sugar moieties on the SGNPs. According to SDS-PAGE and MS of thus obtained proteins, it was found that SGNPs isolated lectins with a high purity. For example, a protein isolated from banana using Glcalpha-GNP (alpha-glucose-immobilized gold nano-particle) was identified as banana lectin by trypsin-digested peptide-MS finger printing method.
Journal of Biochemistry 07/2008; 143(6):833-9. · 2.37 Impact Factor
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ABSTRACT: Carbohydrate chip technology has a great potential for the high-throughput evaluation of carbohydrate-protein interactions. Herein, we report syntheses of novel sulfated oligosaccharides possessing heparin and heparan sulfate partial disaccharide structures, their immobilization on gold-coated chips to prepare array-type Sugar Chips, and evaluation of binding potencies of proteins by surface plasmon resonance (SPR) imaging technology. Sulfated oligosaccharides were efficiently synthesized from glucosamine and uronic acid moieties. Synthesized sulfated oligosaccharides were then easily immobilized on gold-coated chips using previously reported methods. The effectiveness of this analytical method was confirmed in binding experiments between the chips and heparin binding proteins, fibronectin and recombinant human von Willebrand factor A1 domain (rh-vWf-A1), where specific partial structures of heparin or heparan sulfate responsible for binding were identified.
Bioorganic & medicinal chemistry letters 05/2008; 18(7):2499-504. · 2.65 Impact Factor
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ABSTRACT: Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL). In Japan, the number of HTLV-1 carriers is estimated to be 1.2 million and more than 700 cases of ATL have been diagnosed every year. Considering the poor prognosis and lack of curative therapy of ATL, it seems mandatory to establish an effective strategy for the treatment of ATL. In this study, we attempted to identify the cell surface molecules that will become suitable targets of antibodies for anti-ATL therapy. The expression levels of approximately 40,000 host genes of three human T-cell lines carrying HTLV-1 genomes were analyzed by oligonucleotide microarray and compared with the expression levels of the genes in an HTLV-1-negative T-cell line. The HTLV-1-carrying T-cell lines used for experiments had totally different expression patterns of viral genome. Among the genes evaluated, the expression levels of 108 genes were found to be enhanced more than 10-fold in all of the T-cell lines examined and 11 of the 108 genes were considered to generate the proteins expressed on the cell surface. In particular, the CD70 gene was upregulated more than 1,000-fold and the enhanced expression of the CD70 molecule was confirmed by laser flow cytometry for various HTLV-1-carrying T-cell lines and primary CD4(+) T cells isolated from acute-type ATL patients. Such expression was not observed for primary CD4(+) T cells isolated from healthy donors. Since CD70 expression is strictly restricted in normal tissues, such as highly activated T and B cells, CD70 appears to be a potential target for effective antibody therapy against ATL.
Journal of Virology 05/2008; 82(8):3843-52. · 5.40 Impact Factor
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ABSTRACT: A series of ganglioside GM1-, GM2-, and GM3-type probes, in which the ceramide portion is replaced with a glucose residue, were systematically synthesized based on a convergent synthetic method.
Glycoconjugate Journal 05/2008; 25(3):269-78. · 2.12 Impact Factor
