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T Toyokuni

Publications and source records attributed to T Toyokuni.

33 records · Page 2Linked to original sources
PubMed1996

Use of fumonisin B1 to test the intercellular transfer of sphingosine.

Sphingosine, a core unit of sphingolipids, has been shown to mediate intracellular signaling events. It is conceivable that sphingosine could act as an intracellular messenger as well as an intracellular modulator, if sphingosine were to be transferred intercellularly. Murine interleukin-2 (IL-2) dependent T-lymphocyte CTLL cells, murine fibroblasts Swiss 3R3 cells, and murine fibroblast BALB/C A31 cells metabolize exogenously added sphingosine to ceramide. Fumonisin B2, a mycotoxin produced by Fusarium moniliforme, blocks the conversion of sphingosine to ceramide. In the study described here, CTLL cells exhibiting the conversion of sphingosine to ceramide were used as recipient cells, whereas fumonisin B1-treated cells (A31 cells, Swiss 3T3 cells and CTLL cells), in which the conversion was blocked were used as donor cells. To demonstrate intercellular transfer of sphingosine through the conversion pathway of sphingosine to ceramide, fumonisin B1-treated donor cells incorporating radioactive sphingosine were co-incubated with CTLL cells and the ceramide response was examined. These experiments demonstrated that it is possible to prove the intercellular transfer of sphingosine by using different activities in the cellular conversion pathway of sphingosine to ceramide.

3T3 Cells↗
PubMed1994

Further studies on cell adhesion based on Le(x)-Le(x) interaction, with new approaches: embryoglycan aggregation of F9 teratocarcinoma cells, and adhesion of various tumour cells based on Le(x) expression.

We previously proposed specific interaction of Le(x) (Gal beta 1-->4 [Fuc alpha 1-->3]-GlcNAc beta 1-->3Gal) with Le(x) as a basis of cell adhesion in pre-implantation embryos and in aggregation of F9 teratocarcinoma cells, based on several lines of evidence (Eggens et al., J. Biol Chem (1989) 264:9476-9484). We now present additional evidence for this concept, based on autoaggregation studies of plastic beads coated with glycosphingolipids (GSLs) bearing Le(x) or other epitopes, and affinity chromatography on Le(x)-columns of multivalent lactofucopentaose III (Le(x) oligosaccharide) conjugated with lysyllysine. Comparative adhesion studies of Le(x)-expressing tumour cells vs their Le(x)-non-expressing variants showed that only Le(x)-expressing cells adhere to Le(x)-coated plates and are involved in tumour cell aggregation, in analogy to F9 cell aggregation. The major carrier of Le(x) determinant in F9 cells is not GSL but rather polylactosaminoglycan ('embryoglycan'), and we demonstrated autoaggregation of purified embryoglycan in the presence of Ca2+, and reversible dissociation in the absence of Ca2+ (addition of EDTA). Defucosylated embryoglycan did not show autoaggregation under the same conditions. Thus, Le(x)-Le(x) interaction has been demonstrated on a lactosaminoglycan basis as well as a GSL basis. A molecular model of Le(x)-Le(x) interaction based on minimum energy conformation with involvement of Ca2+ is presented.

Carbohydrate Sequence↗
PubMed1994

Synthetic carbohydrate vaccines: synthesis and immunogenicity of Tn antigen conjugates.

A tumor-associated carbohydrate antigen, Tn antigen (GalNAc alpha 1-->O-Ser), was synthesized with a spacer arm, and assembled to dimeric and trimeric structures using N-tert-butyloxycarbonyl-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-alpha- D-galactopyranosyl)-L-serine as a key building block. The synthetic antigens were conjugated with OSA and their immunogenicity examined in mice. Mice immunized with dimeric or trimeric Tn antigen showed a stronger antibody (IgM) response to a Tn-glycoprotein (asialo-ovine submaxillary mucin) than mice immunized with monomeric Tn antigen. The dimeric and trimeric Tn antigens also induced measurable IgG responses. The dimeric Tn antigen was further coupled to a Starburst dendrimer (5th generation) and to tripalmitoyl-S-glycerylcysteinyl-serine, a synthetic lipopeptide of the active moiety of a major lipoprotein of Escherichia coli. Unexpectedly, the Starburst dendrimer conjugate did not stimulate any immune response specific to Tn antigen. On the other hand, immunization of mice with the lipopeptide conjugate produced not only a high IgM response but also significant IgG anti-Tn response without any carrier molecules or additional adjuvants. The production of IgG antibody is quite significant since carbohydrate antigens are in general known to produce only IgM antibody response. Being a totally synthetic, low-molecular weight, and carrier-free immunogen, the lipopeptide conjugate could be a prototype of synthetic carbohydrate vaccines.

Animals↗
PubMed1993

Synthesis of multivalent beta-lactosyl clusters as potential tumor metastasis inhibitors.

A beta-lactosyl residue was linked to the amino groups of L-lysyl-L-lysine through spacer arms of three different lengths (C2, C4, and C9) to give trivalent beta-lactosyl clusters in order to increase the inhibitory activity of the beta-lactosyl group against tumor cell colonization. Thus, O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->4)-2,3, 6-tri-O-acetyl-glucopyranosyl trichloroacetimidate was treated with methyl or benzyl hydroxyethanoate, methyl or benzyl 4-hydroxybutanoate, and methyl 9-hydroxynonanoate, respectively, in the presence of trimethylsilyl trifluoromethanesulfonate to give the corresponding beta-lactosides. These were coupled to L-lysyl-L-lysine, after conversion to the N-hydroxysuccinimide esters, to yield the corresponding trivalent beta-lactosyl-L-lysyl-L-lysine conjugates in good yields. The beta-lactosyl group with a C4 spacer arm was also coupled similarly to poly(L-lysine) (M(r) 3800) to form a polyvalent beta-lactosyl cluster. Coinjection of the trivalent (with C2 and C4 spacer arms) and polyvalent beta-lactosyl clusters with the highly metastatic B16 murine melanoma cells inhibited the formation of lung colonies in C57/BL mice, whereas the trivalent cluster with a C9 spacer arm displayed no activity.

Animals↗
PubMed1991

Chemical engineering of the monoclonal antibody A7 by polyethylene glycol for targeting cancer chemotherapy.

The murine monoclonal antibody A7 (Mab A7) against human colon cancer was chemically modified with methoxypolyethylene glycol (PEG) (Mr 5000). A high substitution of PEG molecules on Mab A7 produced a progressive reduction in antibody-binding activity. The pharmacokinetic and immunological properties of PEG-modified monoclonal antibody A7 (Mab A7) and the PEG-modified F(ab')2 fragment, which retained their antibody-binding activity, were assessed and compared with the parent Mab A7 and the parent F(ab')2 fragment. Blood clearance of PEG-modified antibodies appeared to be diminished by PEG modification and was fitted by a two-compartment model. Low PEG-substituted Mab A7 showed less organ uptake in the liver and spleen and similar uptake in the lung and kidney, compared with the parent Mab A7. PEG-F(ab')2 showed less uptake in the liver and kidney. Both preparations exhibited less tissue:blood ratios in all resected organs as compared with parent antibodies. Tumor localization was enhanced by PEG modification for the F(ab')2 fragment, but not by PEG modification for the whole Mab A7. Multiple i.v. administration of PEG-modified antibody to rabbit did not appear to elicit a measurable immune response to the antibody portion of the conjugate. In conclusion, PEG-modified antibodies are promising reagents as drug carriers to the target tumor.

Animals↗
PubMed1990

Polyethylene glycol modification of the monoclonal antibody A7 enhances its tumor localization.

The F(ab')2 fragment of murine monoclonal antibody A7 was covalently bonded to polyethylene glycol (PEG, molecular weight: 5000) and the conjugate was compared to the parent F(ab')2 fragment by in vitro and in vivo studies. PEG-conjugated antibody fragment retained its antigen-binding activity in a competitive radioimmunoassay. The conjugate had a longer half-life and showed increased accumulation in tumors. Although the tumor: blood ratio for parent F(ab')2 fragment was higher than that for the conjugate, it showed higher value than whole MAb A7. The tissue: blood ratios were kept low with the conjugate, indicating that the conjugate was uptaken to normal organ with lesser extent, as compared with parent F(ab')2 fragment. Our findings indicate that this PEG-conjugated F(ab')2 fragment could be a promising carrier for use in targeting cancer chemotherapy.

Animals↗
PubMed1990

A specific enhancing effect of N,N-dimethylsphingosine on epidermal growth factor receptor autophosphorylation. Demonstration of its endogenous occurrence (and the virtual absence of unsubstituted sphingosine) in human epidermoid carcinoma A431 cells.

Our previous study suggested that N,N-dimethylsphingosine, but not unsubstituted sphingosine, could be a modulator of protein kinase C in epidermoid carcinoma A431 cells, since N,N-dimethyl-D-erythrosphingenine showed a stronger stereospecific effect on protein kinase C activity in comparison with N,N-dimethyl-L-erythrosphingenine, unsubstituted D- or L-erythrosphingenine, and gangliosides (Igarashi, Y., Hakomori, S., Toyokuni, T., Dean, B., Fujita, S., Sugimoto, M., Ogawa, T., El-Ghendy, K., and Racker, E. (1989) Biochemistry 28, 6796-6800). Other studies also indicated that commercial sphingosine preparation has an enhancing effect on epidermal growth factor (EGF) receptor kinase activity in A431 cells (Davis, R. J., Girones, N., and Faucher, M. F. (1988) J. Biol. Chem. 263, 5373-5379; Faucher, M. F., Girones, N., Hannun, Y. A., Bell, R. M., and Davis, R. J. (1988) J. Biol. Chem. 263, 5319-5327). In the present paper, we report (i) the effect of N,N-dimethylsphingosine as compared with lyso-glycosphingolipids and other sphingolipid breakdown products on EGF receptor autophosphorylation and (ii) demonstration of endogenous N,N-dimethylsphingosine synthesis and the virtual absence of unsubstituted sphingosine in A431 cells. The autophosphorylation of EGF receptor in the absence of detergent was strongly enhanced by N,N-dimethyl-D-erythrosphingenine; this effect was even obvious in the absence of EGF and synergistic in the presence of EGF. Similar enhancing activity was not produced by N,N-dimethyl-L-erythrosphingenine, D- and L-erythrosphingenine, N-monomethyl-D-erythrosphingenine, N-acetyl-D-erythrosphingenine, or the five lyso-glycosphingolipids tested. Labeling of sphingosine in A431 cells by culturing in medium containing [3H]Ser for various durations, followed by extraction and isolation of sphingolipids by standard procedures, resulted in clear bands corresponding to N,N-dimethylsphingosine and ceramide, whereas the band corresponding to sphingosine was virtually absent. The bands corresponding to N,N-dimethylsphingosine and ceramide intensified when cells were treated with metabolic inhibitor for UDP-Glc:Cer beta-Glc transferase (which causes accumulation of ceramide). These results indicate that N,N-dimethylsphingosine acts as a stereospecific enhancer for EGF receptor kinase and is able to produce EGF-like activity in vitro even in the absence of EGF and detergent. Under physiological conditions, N,N-dimethylsphingosine is the major catabolite resulting from ceramide breakdown.

Carcinoma, Squamous Cell↗
PubMed1990

Effect of lactose derivatives on metastatic potential of B16 melanoma cells.

The effects of various sugars and sugar derivatives on lung colonization (i.e., metastatic deposition) of the highly metastatic BL6 clone of B16 mouse melanoma cells in syngeneic mice were studied, based on the assumption that carbohydrate structures, particularly those with a Gal terminus, play a crucial role in defining the metastatic potential of B16 cells. After incubation with sugar compounds (usually at 0.1 M concentration), tumor cells were injected via the tail vein into 8-week old female mice. Mice were sacrificed after 18-21 days, and tumor cell colonies in lung were counted under a dissecting microscope. Only methyl beta-D-lactoside and lacto-N-tetraose caused significant reduction (35-45% and 36%, respectively) of metastatic deposition compared to controls. Methyl beta-D-lactoside did not exhibit a growth inhibitory effect on BL6 tumor cells, as determined by several methods: in vitro [3H]thymidine incorporation assay, in vitro plating in RPMI-1640 medium culture under physiological conditions followed by cell counting, and in vivo subcutaneous inoculation of age-matched C57/BL mice followed by tumor measurement. These results indicate that the inhibitory effect of methyl beta-D-lactoside on tumor deposition was not related to its effect on tumor cell growth.

Animals↗
PubMed1989

Effect of chemically well-defined sphingosine and its N-methyl derivatives on protein kinase C and src kinase activities.

In view of the possible effects of the sphingoid base on protein kinases, and the fact that the sphingoid bases used in previous studies were not chemically well-defined, we have studied the effects of chemically well-defined sphingosines and their derivatives on kinase activity. Both (4E)-D- and (4E)-L-erythro-sphingenine showed a weak inhibitory effect, and (4E)-L-threo-sphingenine had a moderate inhibitory effect. In contrast, (4E)-N,N-dimethyl-D-erythro-sphingenine and the sphingosine preparation from a commercial source showed a strong inhibitory effect on PK-C in A431 cells as well as on purified PK-C. Synthetic (4E)-D-erythro-sphingenine and several samples of natural sphingosine inhibited v-src or c-src tyrosine kinase activity measured with polyglutamate-tyrosine (4:1) as substrate. N-Acetylated or N-methylated sphingosines did not inhibit src kinase activity, but rather produced a consistent 1.5-2-fold stimulation of such activity.

Animals↗
PubMed1989

Specific interaction between Lex and Lex determinants. A possible basis for cell recognition in preimplantation embryos and in embryonal carcinoma cells.

The Lex determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc-beta 1----R) has been implicated as having a role in mediating compaction of the mouse embryo at the morula stage (Fenderson, B., Zehavi, U., and Hakomori, S. (1984) J. Exp. Med. 160, 1591-1596). Here, we present evidence suggesting a role for Lex in F9 embryonal carcinoma cell adhesion and a mechanism for Lex recognition based on carbohydrate-carbohydrate interaction. Homotypic aggregation of F9 cells was inhibited by lacto-N-fucopentaose III, and F9 cells showed a preferential interaction with Lex liposomes. The following observations suggest that the structure capable of recognizing Lex per se on F9 cells is Lex: (i) Cell surface-labeled components solubilized in octylglucoside, affinity-bound on an Lex-octyl-Sepharose column, contained glycoproteins reactive with anti-Lex antibody. (ii) Liposomes containing Lex showed significant interaction with Lex glycolipid, but not other glycolipids, coated on a plastic surface. (iii) Liposomes containing Lex glycolipid were found to self-aggregate, whereas liposomes containing paragloboside (nLc4) or sialylparagloboside (IV3NeuAcnLc4) did not. (iv) The diffusibility of 3H-labeled lacto-N-fucopentaitol III (but not I or II), incubated with Lex liposome, from the lower to the upper Boyden chamber through a semipermeable membrane was inhibited. In all these experiments (i-iv), the interaction of Lex to Lex (or Lex to lacto-N-fucopentaose III) was clearly observed only in the presence of Ca2+ and Mg2+ and was enhanced by the presence of Mn2+. These interactions were inhibited by EDTA. The results suggest the novel hypothesis that carbohydrate-carbohydrate interactions may play an important role in controlling cell recognition during F9 cell aggregation and during embryonic development.

Animals↗
PubMed1989

A role of carbohydrate-carbohydrate interaction in the process of specific cell recognition during embryogenesis and organogenesis: a preliminary note.

A possible role of cell surface glycoconjugates in cell recognition has been envisioned based on recognition of carbohydrates by cell surface proteins such as endogenous lectins, glycosyltransferases, and hydrolases (refs. 18-22 in text). A new possibility that a specific carbohydrate at the cell surface could be recognized by the same or similar carbohydrate on the counterpart cell surface is now suggested by specific interaction between Lex and Lex, but not between Lex and sialylated or non-substituted type 2 chain. A new hypothesis is hereby proposed for carbohydrate-carbohydrate interactions as recognition signals during embryogenesis and organogenesis.

Antigens, Surface↗
PubMed1988

Synthesis and biological activity of pyochelin, a siderophore of Pseudomonas aeruginosa.

Pyochelin, a phenolic siderophore of Pseudomonas aeruginosa, was synthesized in three steps from salicylonitrile, L-cysteine, and L-N-methylcysteine. The synthetic product was determined to be identical to natural pyochelin by 1H nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, chromatographic analysis, and chemical reactivity with FeCl3 and ammoniacal silver nitrate reagent. Synthetic and natural pyochelin promoted bacterial growth in iron-depleted medium and were also found to mediate iron transport by P. aeruginosa to the same levels. Neopyochelin, a stereoisomeric by-product of the synthesis, showed less biological activity than did pyochelin in iron transport assays.

Iron Chelating Agents↗
PubMedDate not supplied

Imaging transgene expression with radionuclide imaging technologies.

A variety of imaging technologies are being investigated as tools for studying gene expression in living subjects. Noninvasive, repetitive and quantitative imaging of gene expression will help both to facilitate human gene therapy trials and to allow for the study of animal models of molecular and cellular therapy. Radionuclide approaches using single photon emission computed tomography (SPECT) and positron emission tomography (PET) are the most mature of the current imaging technologies and offer many advantages for imaging gene expression compared to optical and magnetic resonance imaging (MRI)-based approaches. These advantages include relatively high sensitivity, full quantitative capability (for PET), and the ability to extend small animal assays directly into clinical human applications. We describe a PET scanner (microPET) designed specifically for studies of small animals. We review "marker/reporter gene" imaging approaches using the herpes simplex type 1 virus thymidine kinase (HSV1-tk) and the dopamine type 2 receptor (D2R) genes. We describe and contrast several radiolabeled probes that can be used with the HSV1-tk reporter gene both for SPECT and for PET imaging. We also describe the advantages/disadvantages of each of the assays developed and discuss future animal and human applications.

Animals↗