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Biomedical subjects

M Ikekita

Publications and source records attributed to M Ikekita.

At least 19 recordsLinked to original sources
PubMed2002

Radioiodination of glycoprotein-conjugated liposomes by using the Bolton-Hunter reagent and biodistribution in tumor-bearing mice.

We have developed a suitable radiolabeling method for our new type of glycoprotein-liposome conjugate (GCL), in order to investigate its potential utility as a drug carrier that can target the cellular functions of carbohydrate-binding proteins. In order to obtain radiolabeled GCL with high labeling efficiency, we introduced p-hydroxyphenylpropyl groups into the liposome membrane through the amine moiety of a constitutive phospholipid, dipalmitoylphosphatidylethanolamine (DPPE) by using Bolton-Hunter reagent (BHR). Radioiodination of the introduced tyrosyl groups was performed by the Chloramine-T method. The labeling efficiency of the BHR-treated liposome conjugate was high in comparison with that of the BHR-untreated liposome conjugate. An in vitro inhibition study showed that the binding affinity of 125I-labeled BHR-treated GCL (125I-F3S-BH) with lectin was twice as high as that of untreated conjugate (125I-F3S). The biodistribution of 125I-F3S-BH in mice was considerably different from that of 125I-F3S. 125I-F3S-BH was more rapidly taken up by the liver and was more rapidly excreted from the liver than 125I-F3S. Moreover, 125I-F3S-BH accumulated more rapidly into the kidneys, which resulted a lower radioactivity in the blood circulation at an earlier time point than in the case of 125I-F3S. The characteristics of tumor accumulation of 125I-F3S-BH and 125I-F3S were similar to those in blood. If F3S is to be employed as an in vivo targeting ligand in biodistribution studies, BHR would be a suitable tool for radiolabeling because it allows GCL to retain the biological activity and characteristics of the unmodified conjugate.

Animals↗
PubMed2001

Structural study of N-linked oligosaccharides of human intercellular adhesion molecule-3 (CD50).

The N-linked oligosaccharides were released from purified human intercellular adhesion molecule (ICAM)-3 by hydrazinolysis. Approximately 6 mol of oligosaccharides were released from 1 mol of ICAM-3. The oligosaccharides reduced with NaB[3H]4 were separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial lectin column chromatography followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endo-glycosidase digestion and by methylation analysis revealed that N-linked oligosaccharides of ICAM-3 are mainly of tri- and tetra-antennary complex-type, about 60% of which contain two to three poly N-acetyllactosamine chains terminated with the type 1 structure and those without the type 1 structure per oligosaccharide. In addition, a small amount of the high mannose-type oligosaccharide with six alpha-mannose residues, which could act as a ligand for the dendritic cell-specific ICAM-3 grabbing nonintegrin, was detected.

Antigens, CD↗
PubMed2001

Establishment of heterotropic liver tissue mass with direct link to the host liver following implantation of hepatocytes transfected with vascular endothelial growth factor gene in mice.

One of the major goals of tissue engineering is to establish an integrated organ in vivo. We have previously shown that transfection of vascular endothelial growth factor (VEGF) gene into hepatocytes promotes tissue formation by engrafted cells. Here we show that tissue growth was significantly enhanced by co-transplantation of hepatocyte growth factor (HGF) and tumor necrosis factor-alpha (TNF alpha) gene transfected hepatocytes with VEGF-gene transfected cells, but tissue islands were scattered nonspecifically in the abdomen of mice. The result brought us forward to the next step to establish an integrated mass and structural formation of liver tissue. We entrapped VEGF gene transfected hepatocytes in a nylon mesh bag and intraperitoneally engrafted close to the liver. Three weeks later, the bag was covered by a thick network of blood vessels, compared to the control. Histological examination showed that the blood vessels penetrated the parenchyma of the engrafted bag and formed a well-developed vessel network in the region. The use of hepatocytes from lacZ transgenic mice and PCR analysis demonstrated survival and albumin production by hepatocytes in the engrafted bag. Our model can potentially be developed into a heterotropic artificial liver with direct access to the host blood circulation.

Actins↗
PubMed2001

Coordinate involvement of cell cycle arrest and apoptosis strengthen the effect of FTY720.

A novel reagent, FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), has been shown to induce a significant decrease of lymphocytes and lymphoma cells and is expected to be a potent immunosuppressant and anti-tumor drug. The decrease in lymphocytes and lymphoma cells is mainly the result of FTY720-induced apoptosis. FTY720 directly affects mitochondria and induces cell death. Moreover, FTY720 activates protein phosphatase (PP) 2A and affects anti-apoptotic intracellular signal transduction proteins to attenuate the anti-apoptotic effect. In this study, we examined the relationship between FTY720-induced apoptosis and cell cycle regulation. FTY720 induced apoptosis significantly at the G0 / G1 phase and caused G0 / G1 cell cycle arrest of the human lymphoma cell lines HL-60RG and Jurkat. Simultaneously, retinoblastoma protein (pRB) was dephosphorylated, suggesting that dephosphorylation of pRB was related to FTY720-induced G0 / G1 cell cycle arrest. Because this dephosphorylation was completely blocked by a specific PP1 / 2A inhibitor, okadaic acid, it appears that FTY720-activated PP2A is essential for FTY720-induced cell cycle arrest. FTY720-induced apoptosis was inhibited by Bcl-2 overexpression in Jurkat cells, but this did not prevent FTY720-induced cell cycle arrest, suggesting that the mechanism of FTY720-induced cell cycle arrest is independent of the mechanism of FTY720-induced apoptosis. These two independent pathways strengthen the effect of FTY720.

Antineoplastic Agents↗
PubMed2000

Immunosuppressant FTY720 induces apoptosis by direct induction of permeability transition and release of cytochrome c from mitochondria.

FTY720 has immunosuppressive activity in experimental organ transplantation and shows a prompt and protracted decrease of blood T lymphocytes upon oral administration. The blood lymphocyte decrease in vivo was mainly a result of FTY720-induced apoptosis. However, this apoptotic mechanism is not well understood. We examined the mechanism of FTY720-induced apoptosis in lymphoma. Western blotting and fluorescent caspase-specific substrate revealed that caspase-3 is involved in FTY720-induced apoptosis, whereas caspase-1 is not. Apoptotic cell death was inhibited by the pan-caspase inhibitor, Z-VAD-FMK, suggesting that caspase activation is essential for FTY720-induced apoptosis. FTY720 reduced mitochondrial transmembrane potential and released cytochrome c from the mitochondria of intact cells as well as in a cell-free system even in the presence of Z-VAD-FMK. As these mitochondrial reactions occurred before caspase activation, we concluded that FTY720 directly influences mitochondrial functions. The inhibition of mitochondrial permeability transition by Bcl-2 overexpression or by chemical inhibitors prevented all apoptotic events occurring in intact cells and in a cell-free system. Moreover, using a cell-free system, FTY720 did not directly affect isolated nuclei or cytosol. These results indicate that FTY720 directly affects mitochondria and triggers permeability transition to induce further apoptotic events.

Apoptosis↗
PubMed2000

Evidence that FTY720 induces T cell apoptosis in vivo.

The immunosuppressant FTY720 induces a drastic decrease in blood lymphocytes, especially T cells; a decrease which is assumed to be the immunosuppressive mechanism of this drug. FTY720 causes cell death in vitro in lymphocytes and leukemia cells. However, the deletion mechanism of blood lymphocytes in vivo remains unclear. We investigated whether administration of FTY720 induced lymphocyte apoptosis in blood circulation. A marked decrease in the number of blood lymphocytes was observed within an hour after a single oral administration of FTY720 at doses of 5-10 mg/kg in rats and mice. Experiments using fluorescein isothiocyanate (FITC)-Annexin V and APO-BRDU methods revealed that FTY720 induced blood lymphocyte apoptosis in a dose-dependent manner. On the other hand, lymphocyte homing to Peyer's patches was proposed as the mechanism underlying the blood lymphocyte decrease at these doses. However, similar results were obtained when using aly/aly mice, which lack Peyer's patches and lymph nodes. Thus, we concluded that apoptosis of blood lymphocytes was induced immediately after administration of FTY720, and the cells could be immediately scavenged by phagocytes or the reticuloendothelial system in addition to Peyer's patches homing. We also concluded that T cells were highly sensitive to FTY720, which induced apoptosis in vivo.

Administration, Oral↗
PubMed2000

Molecular cloning of mouse alpha-1,6-fucosyltransferase and expression of its mRNA in the developing cerebrum.

Complementary DNA encoding mouse alpha-1,6-fucosyltransferase (FUT) was cloned. The deduced primary structure consisted of 575 amino acids and had 96.0% and 93.0% identity with alpha-1,6-FUT of human and porcine origin, respectively. Quantitative analysis of alpha-1,6-FUT mRNA expression during selected developmental stages of the cerebrum showed that the expression increased during later embryonic stages and was highest in the early postnatal stages (P1 to P7), after which it declined somewhat but still remained relatively high in the mature adult. The expression profile suggests important roles of FUT in the developing central nervous system.

Amino Acid Sequence↗
PubMed1999

Developmental changes in Asn-linked neutral oligosaccharides in murine cerebrum.

The changes in Asn-linked oligosaccharide composition in the murine cerebrum during development have been examined by high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS). The oligosaccharides, obtained from murine cerebrum in several developmental stages, were separated by HPLC on anion-exchange and reverse-phase columns. We found that two Asn-linked oligosaccharides, designated oligosaccharide I and oligosaccharide II, had their expression changed during postnatal development. Whereas oligosaccharide I was reduced during brain development, oligosaccharide II was increased. The structures of oligosaccharides I and II were analyzed by ESI-MS and sequential exoglycosidase digestions. Judging from the molecular and fragment ions in each oligosaccharide, the oligosaccharide I was composed of 5Hex+2HexNAc+ABOE (MW 1467.2) and the oligosaccharide II was 3Hex+4HexNAc+DoHex+ABOE (MW 1695.2). The results of sequential exoglycosidase digestion indicated that the oligosaccharide I was an oligomannose type saccharide and the oligosaccharide II was a biantennary complex type saccharide including fucose. The proposed structures are shown below. These results offer an important clue to the role of Asn-linked oligosaccharides associated with development of the central nervous system.

Animals↗
PubMed1998

Strong suppression of feeding by a peptide containing both the nuclear localization sequence of fibroblast growth factor-1 and a cell membrane-permeable sequence.

Earlier studies have shown that fibroblast growth factor (FGF)-1 in the brain regulates feeding behavior. In the present study, food intake in rats was strongly suppressed by an infusion into the lateral cerebroventricle of a synthetic peptide (26 amino acids) which contains both the N-terminal nuclear localization sequence (NLS) of FGF-1 and a recently identified membrane-permeable sequence. When the NLS motif in the peptide was destroyed by mutations of two lysine residues, the mutant peptide failed to affect eating. The results suggest that the NLS of FGF-1 plays an important role in FGF-1-induced feeding suppression and they introduce a novel compound for feeding regulation.

Amino Acid Sequence↗
PubMed1997

Carbohydrate analysis of porcine thyroglobulin isoforms with different iodine contents.

To further validate the relationship between thyroid hormone formation and the carbohydrate structure of thyroglobulin (Tg), we reinvestigated the relationship between the iodine content and the asparagine-linked oligosaccharide structures of porcine Tg. Purified porcine Tg was further separated into isoforms (Tg-F1, -F2 and -F3) with a DEAE-cellulose ion-exchange chromatography column. The iodine residues, neutral sugar and sialic acid were analyzed for the separated Tg isoforms and their asparagine-linked oligosaccharide structures were analyzed. The asparagine-linked oligosaccharides were released from Tg-F1, -F2 and -F3 by hydrazinolysis and each oligosaccharide was labeled with p-aminobenzoic acid octyl ester (ABOE). The ABOE-labeled oligosaccharides from Tg-F1, -F2 and -F3 were analyzed for their relative content in oligosaccharides of each structure type by chemical methods and DEAE- and ConA high-performance liquid chromatography (HPLC) columns. As a result, it was revealed that the Tg fraction eluted at higher ionic strength from a DEAE-cellulose column is apt to contain more of each iodoamino acid, as well as total content of iodine, larger negative zeta-potential, conforming to sialic acid content in the Tg molecule and to a higher content of di-sialo-bi-antennary complex and to high mannose type oligosaccharides. These results support the conclusion that iodine organification of the Tg molecule is correlated with asparagine-linked oligosaccharide completion.

Animals↗
PubMed1993

Benzylideneascorbate induces apoptosis in L929 tumor cells.

Sodium 5,6-benzylideneascorbate (SBA), which is known to be an antitumor substance, was found to induce apoptotic cell death of L929 tumor cells directly in a concentration- and time-dependent manner. The dying cells exhibited cell shrinkage, disappearance of cell surface microvilli, chromatin condensation and DNA fragmentation into nucleosomal oligomers characteristic of apoptosis. These results indicate a possible mechanism by which SBA induces tumor regression in vivo.

Animals↗
PubMed1992

Purification and characterization of the major cationic kallikrein inhibitor in bovine pituitary gland.

The presence of two types of kallikrein inhibitor (cationic and anionic inhibitors) was demonstrated in bovine pituitary gland. These kallikrein inhibitors were separated from the homogenate of bovine posterior pituitary by successive CM-Sephadex chromatography. The major cationic inhibitor was further purified to homogeneity by affinity chromatography using porcine pancreatic beta-kallikrein immobilized on Sepharose 4B and gel filtration. The complete amino acid sequence of this inhibitor was first determined, and it was shown to be a peptide of 58 residues with a calculated molecular weight of 6,511. The Ki value against bovine pituitary kallikrein was 6 x 10(-9) M. The cationic inhibitor was found to be identical with basic pancreatic trypsin inhibitor.

Amino Acid Sequence↗
PubMed1992

Tissue kallikrein clearance by the liver--effect of plasma components on kallikrein uptake by cultured rat hepatocytes.

Rat urinary kallikrein (RUK) injected intravenously to the rat rapidly disappeared from the blood circulation and mainly distributed to the liver. RUK was also incorporated to the primary cultured rat hepatocytes. The amounts of RUK incorporated into the hepatocytes increased by preincubation of RUK with rat plasma. These results and SDS-PAGE analysis suggested some plasma components may be involved in tissue kallikrein clearance in the blood.

Animals↗
PubMed1992

Canine pancreatic kallikrein: enzyme isolation and characterization.

Two forms of canine pancreatic kallikrein, designated as canine pancreatic kallikrein A and B, were separately isolated by ion-exchange, affinity and hydrophobic chromatographies. These enzymes had similar apparent molecular masses, substrate specificities and pH optima. However, kallikrein B was inhibited by soybean trypsin inhibitor, while kallikrein A was not. Both kallikrein A and B were shown by sodium dodecyl sulfate-polyacryl amide gel electrophoresis to consist of two polypeptide chains, designated alpha and beta chains, and binding by disulfide bond(s). The N-terminal amino acid sequences of each alpha and beta chains of kallikrein A and B were determined.

Amino Acid Sequence↗
PubMed1990

Observation of tissue prokallikrein activation by some serine proteases, arginine esterases in rat submandibular gland.

Two serine proteases, arginine esterases (esterases I and II) which showed the activity of tissue prokallikrein activation were identified in rat submandibular gland. These enzymes were separated from the homogenate of rat submandibular gland by two successive DEAE-cellulose chromatographies and were further purified and characterized. Esterases I and II were found to be identical with tonin and esterase B-like enzyme, respectively. Both enzymes activated rat urinary prokallikrein at near neutral pH. Esterase B-like enzyme activated rat urinary prokallikrein better than tonin.

Amino Acid Sequence↗