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Introduction Syncytiotrophoblasts are the major components of the human placenta involved in fetal maternal exchange and hormone secretion. The syncytiotrophoblasts arise from the fusion of villous cytotrophoblasts. The cell cycle suppressor p57KIP2 is known to be an essential molecule for proper trophoblast differentiation during placental formation. Methods We generated p57KIP2-expressing BeWo transfectant cells. Proliferation assay and matrigel invasion assay were used to characterize p57KIP2-expressing BeWo transfectant cells. To reveal the role of p57KIP2 in syncytialization, we proceeded syncytium formation analysis and qRT-PCR for detection of the expression levels Syncytin-1, Syncytin-2 and their receptors. Results The human choriocarcinoma cell line, BeWo has undetectable levels of p57KIP2 expression. Expression of p57KIP2 reduced cell proliferation rate and extracellular matrix invasion activity. p57KIP2 expressing cells displayed multinucleated cells associated with syncytiotrophoblast differentiation. In the syncytialization event, p57KIP2 was found to potentiate forskolin-induced upregulation of Syncytin-2 in a cAMP-independent manner. Discussion These results indicate that the expression of p57KIP2 may act on the proliferation/invasion inhibitory factor and enhance the expression of Syncytin-2, which are associated with syncytialization in cytotrophoblasts.

We examined whether chondroitin sulfates (CSs) exert inhibitory effects on heparanase (Hpse), the sole endoglycosidase that cleaves heparan sulfate (HS) and heparin, which also stimulates chemokine production. Hpse-mediated degradation of HS was suppressed in the presence of glycosaminoglycans derived from a squid cartilage and mouse bone marrow-derived mast cells, including the E unit of CS. Pretreatment of the chondroitin sulfate E (CS-E) with chondroitinase ABC abolished the inhibitory effect. Recombinant proteins that mimic pro-form and mature-form Hpse bound to the immobilized CS-E. Cellular responses as a result of Hpse-mediated binding, namely, uptake of Hpse by mast cells and Hpse-induced release of chemokine CCL2 from colon carcinoma cells, were also blocked by the CS-E. CS-E may regulate endogenous Hpse-mediated cellular functions by inhibiting enzymatic activity and binding to the cell surface.

Leukocyte migration is essential for exerting self-defense mechanisms. During the extravasation process, leukocytes transmigrate through the endothelial lining and the subendothelial basement membrane. Accumulating evidence supports the involvement of heparanase in this process. Altered cellular distribution resulting in relocalization of heparanase to the leading edge of migration is a key event to rapidly turn on the function of the enzyme during migration. This review presents current research investigating the cellular machinery that builds up a functional subcellular structure for leukocyte attachment to and degradation of the extracellular matrix. Recent advances in the understanding of the roles of heparanase in inflammatory diseases and pharmacological approaches to control heparanase-mediated actions during inflammation are also discussed.

Microbial metabolites have attracted increasing interest as a source of therapeutics and as probes for biological mechanisms. New microbial metabolites and derivatives targeted at inflammation and bone disease therapy have been identified by focusing on prostaglandin release, osteoblast differentiation and immune cell functions. These modulators of inflammatory processes and bone disease contribute to our understanding of biological mechanisms and support identification of the therapeutic potential of drug lead candidates. The present review describes recent advances in the chemistry and analysis of inhibitors of prostaglandin release or other functional molecules of immune cells, as well as inducers of osteoblast differentiation, including biological and pharmacological activities.The Journal of Antibiotics advance online publication, 1 November 2017; doi:10.1038/ja.2017.138.

To explore possible roles of heparanase in cancer-host crosstalk, we examined whether heparanase influences expression of inflammatory chemokines in colorectal cancer cells. Murine colorectal carcinoma cells incubated with heparanase upregulated MCP-1, KC, and RANTES genes and released MCP-1 and KC proteins. Heparanase-dependent production of IL-8 was detected in two human colorectal carcinoma cell lines. Addition of a heparanase inhibitor Heparastatin (SF4) did not influence MCP-1 production, while both latent and mature forms of heparanase augmented MCP-1 release, suggesting that heparanase catalytic activity was dispensable for MCP-1 production. In contrast, addition of heparin to the medium suppressed MCP-1 release in a dose-dependent manner. Similarly, targeted suppression of Ext1 by RNAi significantly suppressed cell surface expression of heparan sulfate and MCP-1 production in colon 26 cells. Taken together, it is concluded that colon 26 cells transduce the heparanase-mediated signal through heparan sulfate binding. We propose a novel function for heparanase independent of its endoglycosidase activity, namely as a stimulant for chemokine production.

We investigated the fate of proheparanase added to the culture media of mast cells. A recombinant protein mimicking proheparanase was continuously internalized into mastocytoma cells as well as bone marrow- and peritoneal cell-derived mast cells. Internalized heparanase molecules were accumulated in granules and a significant portion was released by stimulation with ionomycin, indicating that the internalized heparanase was sorted into secretory granules. The pro-form heparanase was processed into a mature and an active form inside the cells, in which intracellular heparin was fragmented by the mature enzyme. The internalization was substantially inhibited by addition of heparin and heparan sulfate to the culture medium, suggesting that glycosaminoglycan is involved in the uptake pathway. Out of four syndecans, expression of syndecan-3 and syndecan-4, especially cell surface syndecan-4, was detected in the mastocytoma cells. Two knockdown clones transfected with a shRNA expression vector targeting the syndecan-4 gene took up significantly lower amounts of heparanase than mock cells. We propose that some exogenous substances like proheparanase can be incorporated into mast cell granules via a glycosaminoglycan-mediated, especially syndecan-4-dependent, uptake pathway.

Protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) is a Golgi glycosyltransferase that catalyzes the formation of the N-acetylglucosamine (GlcNAc) β1→2Man linkage of O-mannosyl glycan. POMGNT1 is not modified by N-glycans because there are no potential N-glycosylation sites; however, it is not clear whether POMGNT1 is modified by O-glycans. To determine whether POMGNT1 is O-glycosylated, we prepared recombinant human POMGNT1 from HEK293T cells. The recombinant POMGNT1 was recognized by Sambucus sieboldiana lectin (SSA), and sialidase digestion of POMGNT1 decreased SSA reactivity and enhanced the reactivity of Arachis hypogaea lectin (PNA). These results suggest that POMGNT1 is modified by a sialylated core-1 O-glycan. Next, we analyzed the structures of the O-glycans on POMGNT1 by β-elimination and pyrazolone-labeling methods in combination with mass spectrometry. We identified several mucin-type O-glycans containing (NeuAc)1(Hex)1(HexNAc)1, (NeuAc)2(Hex)1(HexNAc)1, and (NeuAc)2(Hex)2(HexNAc)2. To examine whether the O-glycans affect the functions and properties of POMGNT1, we compared glycosylated and non-glycosylated forms of recombinant sPOMGNT1 for their activity and surface hydrophobicity using the hydrophobic probe 1-anilino-8-naphthalene sulfonate (ANS). POMGNT1 activity and surface hydrophobicity were not affected by the presence or absence of O-glycans.

Heparanase cleaves macromolecular heparin in the secretory granules of connective tissue-type mast cells. We investigated roles of the cleavage under a microenvironment mimicking where the mast cells physiologically reside. A connective tissue-type mast cell line MST and mouse peritoneal cell-derived mast cells stored macromolecular heparin in the secretory granules. The cells expressing heparanase stored fragmented heparin (~10 kDa) due to heparanase-dependent cleavage of the heparin. We produced an artificial collagen-based extracellular matrix and placed the live cells or glycosaminoglycans purified from the cells in the matrix to measure the release of sulfated macromolecules into the medium. The sulfate-radiolabelled molecules from the degranulating heparanase-expressing cells and the purified glycosaminoglycans showed significantly greater release into the medium than those derived from mock cells, which was not the case in suspension culture. The mast cell granular enzyme chymase, but not β-hexosaminidase, showed significantly greater release from the degranulating heparanase-expressing cells than from mock cells. Purified chymase mixed with fragmented heparin derived from heparanase-expressing cells showed greater release from collagen gels than the enzyme alone or mixed with macromolecular heparin derived from mock cells. We propose that the cleavage of macromolecular heparin by heparanase accelerates the release of heparin and chymase from extracellular matrices.

Local infiltration of inflammatory cells is regulated by a number of biological steps during which the cells likely penetrate through subendothelial basement membranes that contain heparan sulfate proteoglycans. In the present study, we examined whether administration of heparastatin (SF4), an iminosugar-based inhibitor of heparanase, could suppress local inflammation and degradation of heparan sulfate proteoglycans in basement membranes. In a carrageenan- or formyl peptide-induced dorsal air pouch inflammation model, the number of infiltrated neutrophils and monocytes was significantly lower in mice after topical administration of heparastatin (SF4). The concentration of chemokines MIP-2 and KC in pouch exudates of drug-treated mice was similar to control. In a zymosan-induced peritonitis model, the number of infiltrated cells was not altered in drug-treated mice. To further test how heparastatin (SF4) influences transmigration of inflammatory neutrophils, its suppressive effect on migration and matrix degradation was examined in vitro. In the presence of heparastatin (SF4), the number of neutrophils that infiltrated across a Matrigel-coated polycarbonate membrane was significantly lower, while the number of neutrophils passing through an uncoated membrane was not altered. Lysate of bone marrow-derived neutrophils released sulfate-radiolabeled macromolecules from basement membrane-like extracellular matrix, which was suppressed by heparastatin (SF4). Heparan sulfate degradation activity was almost completely abolished after incubation of lysate with protein G-conjugated anti-heparanase monoclonal antibody, strongly suggesting that the activity was due to heparanase-mediated degradation. Taken together, in a dorsal air pouch inflammation model heparastatin (SF4) potentially suppresses extravasation of inflammatory cells by impairing the degradation of basement membrane heparan sulfate.

The authors recently demonstrated that the mitochondrial voltage-dependent anion channel 1 (VDAC1) is involved in the sensitivity of cancer cells to furanonaphthoquinone (FNQ). The aim of the present study was to investigate whether mitochondrial VDAC1 reduces quinone antitumor drugs. The VDAC1 purified by immunoprecipitation reduced FNQ in the presence of nicotinamide adenine dinucleotide (NADH) and produced H(2)O(2). Blue native polyacrylamide gel electrophoresis demonstrated that the band that reduced FNQ NADH-dependently mainly included VDAC1. Because H(2)O(2) generation in catalyzing FNQ with NADH caused mitochondrial damage, the cytotoxic activity of FNQ was induced by VDAC1. In the quinone antitumor drugs, menadione (VK3), adriamycin and mitomycin C, mitochondrial VDAC1 bioreductively activated VK3. These results demonstrate that mitochondrial VDAC1 is a pharmacologic target for the treatment of tumor.

The role of a macrophage galactose-type calcium-type lectin-1 (MGL1) in antigen-independent granulation tissue formation was investigated. Granulation tissue was induced by injection of carrageenan in an air pouch and distribution of macrophages expressing MGL1/2 was histologically examined. MGL1/2-positive cells were not observed in the granulation tissue induced by carrageenan though these cells were present in dermis. This was distinct from the fact that MGL1/2-positive cells were abundant in granulation tissue induced by antigenic stimulation. CD11b-positive cells were in dermis and carrageenan-induced granulation tissue. Because antigen-induced granulation tissue formation was previously shown to decrease in MGL1-deficient mice or after treatment with anti-MGL1 antibody, we investigated the effects of MGL1-deficient status on carrageenan-induced granulation tissue formation. The thickness of granulation tissue was almost identical between wild-type and MGL1-deficient mice. It is highly likely that MGL1-positive cells are not involved in tissue remodeling when inflammation is driven by nonspecific stimuli.

In the sensitization phase of delayed-type hypersensitivity, dermal macrophages (MOs) expressing MO galactose-type C-type lectin1 (MGL1) are known to migrate from the dermis to lymph nodes (LNs), and this migration was previously suggested to be an essential process in the establishment of the immune response to peripheral antigens. We hypothesize that the interactions between MGL1 and its ligands determine the localization of MGL1/2-positive cells within the LNs. In the present report, distribution of MGL1 binding sites was immunohistochemically examined by means of biotinylated recombinant MGL1 and compared with the distribution of MGL1/2-positive cells after epicutaneous sensitization of the mice with fluorescein isothiocyanate. The binding sites of MGL1 were mainly detected on the subcapsular sinus, interfollicular regions, and the T/B boundary. The distribution overlapped with the area where MGL1/2-positive cells that had migrated from dermis localized after sensitization with the antigen.

Filoviruses cause lethal hemorrhagic disease in humans and nonhuman primates. An initial target of filovirus infection is the mononuclear phagocytic cell. Calcium-dependent (C-type) lectins such as dendritic cell- or liver/lymph node-specific ICAM-3 grabbing nonintegrin (DC-SIGN or L-SIGN, respectively), as well as the hepatic asialoglycoprotein receptor, bind to Ebola or Marburg virus glycoprotein (GP) and enhance the infectivity of these viruses in vitro. Here, we demonstrate that a recently identified human macrophage galactose- andN-acetylgalactosamine-specific C-type lectin (hMGL), whose ligand specificity differs from DC-SIGN and L-SIGN, also enhances the infectivity of filoviruses. This enhancement was substantially weaker for the Reston and Marburg viruses than for the highly pathogenic Zaire virus. We also show that the heavily glycosylated, mucin-like domain on the filovirus GP is required for efficient interaction with this lectin. Furthermore, hMGL, like DC-SIGN and L-SIGN, is present on cells known to be major targets of filoviruses (i.e., macrophages and dendritic cells), suggesting a role for these C-type lectins in viral replication in vivo. We propose that filoviruses use different C-type lectins to gain cellular entry, depending on the cell type, and promote efficient viral replication.

Lectins on antigen presenting cells are potentially involved in the antigen uptake and the cellular recognition and trafficking. Serial analysis of gene expression in monocyte-derived dendritic cells (DCs), monocytes, and macrophages revealed that 7 of the 19 C-type lectin mRNA were present in immature DCs. Two of these, the macrophage mannose receptor and the macrophage lectin specific for galactose/N-acetylgalactosamine (MGL), were found only in immature DCs, as confirmed by reverse transcriptase-PCR and flow cytometric analysis. By subcloning and sequencing the amplified mRNA, we obtained nucleotide sequences encoding seven different human MGL (hMGL) subtypes, which were apparently derived from alternatively spliced mRNA. In addition, the hMGL gene locus on human chromosome 17p13 contains one gene. A single nucleotide polymorphism was identified at a position in exon 3 that corresponds to the cytoplasmic region proximal to the transmembrane domain. Of all the splicing variants, the hMGL variant 6C was expressed at the highest levels on immature DCs from all donors tested. Immature DCs could incorporate alpha-GalNAc-modified soluble acrylamide polymers, and this was significantly inhibited by pretreatment of the cells with an anti-hMGL monoclonal antibody that blocks the lectin-carbohydrate interaction. We propose that hMGL is a marker of imDCs and that it functions as an endocytic receptor for glycosylated antigens.

Cytotoxic properties of human activated macrophages were investigated by measuring both 51 Cr and [ 3 H]TdR releases from prelabeled target cells. The kinetic study of macrophage-mediated cytotoxicity indicated that 51 Cr and [ 3 H]TdR releases showed a distinct time course. Next, when [ 3 H]TdR release was measured as a parameter for cytotoxicity, pretreatment of activated macrophages with either actinomycin D or emetine remarkably decreased their cytotoxicity in a dose-dependent manner. In contrast, the pretreatment with these inhibitors had little effect on 51 Cr release. Furthermore, the addition of Zn 2+ in the assay medium caused the decrease in [ 3 H]TdR release but not 51 Cr release from the target cells. Taken together, our findings indicate that 51 Cr and [ 3 H]TdR releases from the target cells are induced by activated macrophages through different cytotoxic mechanisms; the former does not require RNA and protein syntheses in macrophages during coculture, whereas the latter requires them. Our findings also suggest that macrophage-derived nuclease may play an important role for inducing [ 3 H]TdR release from the target cells.

The tandem use of the photosensitive bola-amphiphile 1 (X=3 H) and cholesterol enabled the determination of the center of the transmembrane domain of glycophorin A (131 amino acid residues) in a membrane by selective functionalization of the protein within a phospholipid bilayer.

O-glycosylation of mucin is initiated by the attachment of N-acetyl-D-galactosamine (GalNAc) to serine or threonine residues in mucin core polypeptides by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). It is not well understood how GalNAc attachment is regulated by multiple ppGalNAc-Ts in each cell. In the present study, the expression levels of murine ppGalNAc-Ts (mGalNAc-Ts), T1, T2, T3, T4, T6, and T7 were compared between mouse colon carcinoma colon 38 cells and variant SL4 cells, selected for their metastatic potentials, by using the competitive RT-PCR method. The expression levels of mGalNAc-T1, T2, and T7 were slightly higher in the SL4 cells than in the colon 38 cells, whereas the expression level of mGalNAc-T3 in the SL4 cells was 1.5% of that in the colon 38 cells. Products of enzymatic incorporations of GalNAc residues into FITC-PTTTPITTTTK peptide by the use of microsome fractions of these cells as the enzyme source were separated and characterized for the number of attached GalNAc residues and their positions. The maximum number of attached GalNAc residues was 6 and 4 when the microsome fractions of the colon 38 cells and SL4 cells were used, respectively. When the microsome fractions of the colon 38 cells were treated with a polyclonal antibody raised against mGalNAc-T3, the maximum number of incorporated GalNAc residues was 4. These results strongly suggest that mGalNAc-T3 in colon 38 cells is involved in additional transfer of GalNAc residues to this peptide.

We compared the tumoricidal activity of human monocytes cultured with interleukin-2 (IL-2) or human recombinant interferon-gamma (IFN-gamma) alone, or IFN-gamma in combination with a small amount of lipopolysaccharides (LPS). Human monocytes cultured with IL-2 for 7 days or longer, termed lymphokine-activated macrophages (LAMs), showed higher tumoricidal activity than those cultured for 1 day. In contrast, monocytes cultured with IFN-gamma alone or in combination with LPS for 7 days or longer showed lower tumoricidal activity. LAMs were identified as macrophages by nonspecific esterase staining and immunofluorescence staining with anti-CD14 antibody. LAMs were not induced in fetal calf serum-containing medium, but they were induced when colony-stimulating factor-1 was added to the medium. LAMs showed high tumoricidal activity against all human and murine tumor cell lines tested, although they showed no cytotoxic activity against human normal cells. During incubation with IL-2, tumoricidal activity of LAMs was maximal at days 8-16 and was sustained until day 28. The difference in tumoricidal mechanism between LAMs and lymphokine-activated killer (LAK) cells was also shown by using two kinds of cytotoxic assay systems. LAMs require a long incubation time to kill tumor cells, but LAK cells can kill them immediately. Furthermore, LAMs kill tumor cells with complete DNA degradation, whereas LAK cells can induce significant but not complete DNA degradation. These results indicate that LAMs and LAK cells have different tumoricidal mechanisms for killing target cells, although they were induced by incubation with the same lymphokine, IL-2.

In the sensitization phase of contact hypersensitivity in mice, dermal macrophages (MOs) expressing MO galactose-type C-type lectin1 (MGL1) are known to migrate from the dermis to lymph nodes (LNs) where they accumulate in the subcapsular sinus, interfollicular regions, and areas surrounding high endothelial venules. We hypothesize that the interactions between MGL1 and its ligands determine the localizations of MGL1-positive cells within the LNs. In the present study, our major aim was to isolate MGL1 counter-receptor(s) from lysates of LNs using affinity chromatography with immobilized recombinant MGL1. Fractions bound and eluted with EDTA were analyzed by SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. One of the predominant components was sialoadhesin (Sn, Siglec-1). Sn from lysates of LNs was immobilized on microtiter plates precoated with anti-Sn monoclonal antibody, and binding of recombinant MGL1 and adhesion of cells expressing MGL1 were tested. The binding of recombinant MGL1 to Sn was shown to be dependent on Ca2+ and N-glycans on Sn. MGL1-transfected Chinese hamster ovary cells adhered to the Sn-coated plates, whereas mock transfectants did not. Immunohistochemical localization of anti-Sn monoclonal antibody in LN coincided with the subcapsular sinus area to which recombinant MGL1 was bound. Furthermore, the distribution of MGL1+ cells after sensitization with FITC was demonstrated to overlap with that of Sn within the subcapsular sinus of draining LNs. These results suggest that Sn acts as an endogenous counter-receptor for MGL1.

Extravasation of peripheral blood monocytes through vascular basement membranes requires degradation of extracellular matrix components including heparan sulfate proteoglycans (HSPGs). Heparanase, the heparan sulfate-specific endo-β-glucuronidase, has previously been shown to be a key enzyme in melanoma invasion, yet its involvement in monocyte extravasation has not been elucidated. We examined a potential regulatory mechanism of heparanase in HSPG degradation and transmigration through basement membranes in leukocyte trafficking using human promonocytic leukemia U937 and THP-1 cells. PMA-treated cells were shown to degrade 35S-sulfated HSPG in endothelial extracellular matrix into fragments of an approximate molecular mass of 5 kDa. This was not found with untreated cells. The gene expression levels of heparanase or the enzyme activity of the amount of cell lysates were no different between untreated and treated cells. Immunocytochemical staining with anti-heparanase mAb revealed pericellular distribution of heparanase in PMA-treated cells but not in untreated cells. Cell surface heparanase capped into a restricted area on PMA-treated cells when they were allowed to adhere. Addition of a chemoattractant fMLP induced polarization of the PMA-treated cells and heparanase redistribution at the leading edge of migration. Therefore a major regulatory process of heparanase activity in the cells seems to be surface expression and capping of the enzyme. Addition of the anti-heparanase Ab significantly inhibited enzymatic activity and transmigration of the PMA-treated cells, suggesting that the cell surface redistribution of heparanase is involved in monocyte extravasation through basement membranes.

Egg PC liposome as reconstituted with poly(ethylene oxide)-bearing lipid (coded as PEO-lipid (n = 15)) was remarkably endocytosed by Jurkat cell, which was a lymphoblastoma derived from human T cell. To confirm the endocytosis, two kinds of fluorescent probes (FITC-dextran and octadecyl rhodamine B) were employed. The former was loaded in the aqueous phase of the liposome, while the latter was embedded in the liposomal membrane. Both probes were found coincidentally at the same site in the cytosol, clearly suggesting that whole liposome entered the cell. The endocytosis was most obvious when PEO-lipid (n = 15) was employed above 50 mol%. FITC-Dextran entered the cell was found small dots in the cell, not dispersive. Even when octadecyl rhodamine B was used, no membrane fluorescence was observed at all. The uptake closely related to the cell metabolism as affected by the culture temperature and serum in the incubation medium. Furthermore, the addition of cytochalasin B completely prohibited the cell uptake of liposomes.

We have developed a chemical method for directly identifying the amino acid residues of the transmembrane domain of a protein that are located right in the center of the membrane. Glycophorin A (GPA), the major sialoglycoprotein of human erythrocytes, was the first membrane protein whose primary sequence was elucidated, but its three-dimensional structure is still not known. GPA has been reconstituted into liposomes formed from dimyristoylphosphatidylcholine, dimyristoylphosphatidylserine, cholesterol, and a bola-amphiphilic phospholipidic photoactivatable probe (radioactive probe 1) by a detergent-mediated method. Electron microscopy confirmed the formation of spherical vesicular structures, and sucrose-density gradients revealed that the proteoliposomes comprised only one membrane fraction. Proteinase-K digestion of GPA in the proteoliposomes suggested that the orientation of GPA in reconstituted proteoliposomes was virtually identical to that observed in natural erythrocyte membranes. After photo-irradiation of the reconstituted proteoliposomes and in situ tryptic digestion, the photolabeled amino acid residues were analyzed by Edman degradation and their radioactivity was measured. Val80 and Met81, which had been assumed to be located near the center of the transmembrane domain of GPA, were indeed highly selectively photolabeled by probe 1. The new method might be applied to analyze the three-dimensional arrangement of the transmembrane domain of protein complexes that are made up from several subunits.

Direct fusion between Jurkat cell and a liposome modified with poly(ethylene oxide)-bearing lipid (PEO-lipid) was examined using diphtheria toxin fragment A (DTA) as the probe. Only the DTA-loaded liposome modified with PEO-lipid(n = 32) (n is the number of ethylene oxide units) exerted significant cytotoxicity against Jurkat cells, while liposomes lacking either the PEO-lipid or DTA did not. Liposomes modified by the PEO-lipid with shorter PEO chain(n = 5 or 15) did not show any cytotoxicity, irrespective of their DTA-loading. The cytotoxicity was observed even in the presence of cytochalasin B, an inhibitor of endocytosis. Judging from these results, we concluded that the PEO-lipid(n = 32)-modified liposome directly fused with plasma membrane of Jurkat cell.