Biomedical subjects

T Takagi

Publications and source records attributed to T Takagi.

At least 19 recordsLinked to original sources

PubMed1992

Nucleotide sequence of a complementary DNA for human ST2.

Human ST2 cDNA, a homologue of murine ST2 that is only expressed in growth-stimulated BALB/c-3T3 cells and a member of the primary response gene family induced by growth factors, was isolated from the cDNA library of an activated human helper T cell line, 5C10. Human ST2 has 67.6% identity in a 327 amino acid overlap to murine ST2. Furthermore, as in the case of murine ST2, human ST2 encodes a protein remarkably similar in sequence to the extracellular portion of human interleukin 1 receptor, both types 1 and 2. The expression of ST2 in human lymphocytes could trigger further investigations into its physiological role in humans.

3T3 Cells↗

PubMed1992

Role of liver-type glucose transporter (GLUT2) in transport across the basolateral membrane in rat jejunum.

To obtain information on the regulation of glucose transport across the basolateral membrane (BLM) of intestinal epithelial cells, we measured the number of [3H]cytochalasin B binding sites and the level of liver-type glucose transporter (GLUT2) protein in the BLM in the jejunum of rats (i) with diabetes (ii) given a high-carbohydrate diet or (iii) with experimental hyperglycemia (12 h infusion of a high-glucose solution). A glucose uptake and the number of D-glucose inhibitable [3H]cytochalasin B binding sites in BLM vesicles were significantly increased in all three conditions. Western blot analysis showed that the amount of GLUT2 protein in BLM vesicles was increased in rats with diabetes and those given a high-carbohydrate diet, but not in those with experimental hyperglycemia. These results suggest that there is a mechanism for rapid regulation of glucose transport in the BLM that does not depend on change in the amount of GLUT2.

Animals↗

PubMed1992

A myoglobin evolved from indoleamine 2,3-dioxygenase.

Hemoglobins and myoglobins are some of the best studied proteins. They are distributed in animals, plants and bacteria, and the characteristic two intron-three exon structure is widely conserved in animal globin genes (Jhiang et al., 1988). To date, all of the hemoglobins and myoglobins are believed to have a common origin, and so they are considered to be homologous. We have isolated a completely new type of myoglobin from the red muscle of the abalone Sulculus diversicolor aquatilis. The myoglobin consists of an unusual 41 kDa polypeptide chain, contains one heme per chain and forms a homodimer under physiological conditions. The cDNA-derived amino acid sequence of Sulculus myoglobin showed no significant homology with any other globins, but, surprisingly, showed high homology (35% identity) with human indoleamine 2,3-dioxygenase, a tryptophan degrading enzyme containing heme. This clearly indicates that Sulculus myoglobin evolved from a gene for indoleamine dioxygenase, but not from a globin gene. Sulculus myoglobin lacks the enzyme activity of indoleamine dioxygenase. However, in the presence of tryptophan, the autoxidation rate of oxymyoglobin was greatly accelerated, suggesting that a tryptophan binding site remains near or in the heme cavity as a relic of the molecular evolution.

Amino Acid Sequence↗

PubMed1992

Amino acid sequence of yeast hemoglobin. A two-domain structure.

The complete amino acid sequence of a hemoglobin from yeast (Candida norvegensis) has been determined by peptide and cDNA sequence analyses. The protein is composed of 387 amino acid residues and its amino terminus was blocked by an acetyl group. A computer search showed that the sequence of 155 N-terminal residues has 39% homology with that of Vitreoscilla hemoglobin. On the other hand, the sequence of 230 C-terminal residues showed a small, but notable, degree of similarity with that of a methemoglobin reductase found in human erythrocyte, i.e. NADH-cytochrome b5 oxido-reductase. We therefore conclude that yeast hemoglobin consists of two distinct domains; one is a heme-containing oxygen binding domain of the N-terminal region and the other is an FAD-containing reductase domain found in the C-terminal region.

Amino Acid Sequence↗

PubMed1992

Calmodulin cDNAs from two species of Tetrahymena.

We describe the isolation and characterization of cDNAs encoding calmodulins of Tetrahymena thermophila and Tetrahymena pyriformis. It reveals that the deduced amino acid sequences of both calmodulins are precisely the same.

Amino Acid Sequence↗

PubMed1992

Molecular weight determination and compositional analysis of dextran-protein conjugates using low-angle laser light scattering technique combined with high-performance gel chromatography.

The low-angle laser light scattering technique combined with high-performance gel chromatography was applied for characterization of the dextran-ovalbumin and dextran-lysozyme conjugates obtained from the mild heating in dry state, which is attracting interest as a way leading to stabilization of proteins and to production of proteins with excellent emulsifying or antimicrobial ability (Nakamura, S., Kato, A. and Kobayashi, K. (1991) J. Agric. Food Chem. 39, 647-650). According to the above technique, providing the information about the molecular weight distribution and the composition of the conjugates, one or two dextran molecules were found to be linked to one molecule of the proteins. In addition, each of the conjugates was shown to exist as an oligomeric assembly of which formation is promoted by an increase in the salt concentration of buffer. The observations suggest that the increase in the hydrophobicity of the protein moiety as a result of partial denaturation and the introduction of the hydrophilic dextran chain affords the conjugate an amphiphilic property.

Chromatography, Gel↗

PubMed1992

High-performance hydroxyapatite chromatography of integral membrane proteins and water-soluble proteins in complex with sodium dodecyl sulphate.

Integral membrane proteins from human erythrocytes were fractionated in the presence of sodium dodecyl sulphate (SDS) on four types of high-performance hydroxyapatite columns. A column of 2-microns sintered hydroxyapatite beads from Asahi Optical (Tokyo, Japan) gave the best resolution. With this column, glycophorin was eluted early in a gradient of increasing sodium phosphate buffer concentration, the glucose transporter was eluted later in two zones, one of which contained this protein alone, and the anion transporter was eluted last. Water-soluble proteins applied in complex with SDS also separated reasonably well upon elution. The water-soluble proteins and the membrane proteins were all eluted mainly in the order of increasing polypeptide length, but with considerable individual variation. SDS-polypeptide complexes are probably adsorbed onto hydroxyapatite by the interaction of positively charged amino acid side groups with phosphate ions (at P-sites) and of negatively charged amino acid side groups and polypeptide-bound dodecyl sulphate anions with calcium ions (at C-sites). As a rule, the number of charged side groups and dodecyl sulphate anions, and thus the number of binding sites, increases with the polypeptide chain length, which explains the general order of release of the polypeptides.

Amino Acids↗

PubMed1992

Coexpression of the receptor-associated protein gephyrin changes the ligand binding affinities of alpha 2 glycine receptors.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein, whose ligand binding alpha subunit occurs in several isoforms in the mammalian central nervous system. Here we show that coexpression of the GlyR-associated protein gephyrin changes the agonist and antagonist binding affinities of GlyRs generated by alpha 2 subunit expression in 293 kidney cells. Thus, a receptor-associated protein modifies the functional properties of a neurotransmitter receptor. This may contribute to an optimization of the postsynaptic neurotransmitter response.

Animals↗

PubMed1992

Molecular weights of glycosylated and nonglycosylated forms of recombinant human stem cell factor determined by low-angle laser light scattering.

The molecular weight of recombinant human stem cell factor (SCF) was determined using a low-angle laser light scattering combined with a differential refractometer and a uv detector. The protein samples were applied to these detectors through a gel filtration column by a high-performance liquid chromatographic pump. The Chinese hamster ovary (CHO) cell-derived SCF gave a molecular weight of 53,000 for the entire molecule and 35,000 for the protein moiety only at pH 7.0, indicating that the CHO cell-derived protein is glycosylated by 34%. Since the molecular weight of the polypeptide is 18,600, the results demonstrate that the CHO cell-derived SCF forms a dimer. The molecular weight of Escherichia coli-derived SCF was determined to be 39,000, similar to the above value (35,000). Essentially identical molecular weights were obtained at pH 3.0, indicating no dissociation of the dimer.

Animals↗

PubMed1992

Primary structure of three minor isoforms of amphioxus sarcoplasmic calcium-binding proteins.

Previously we reported the amino acid sequences of 4 well-defined sacroplasmic, high-affinity Ca(2+)-binding proteins in the protochordate amphioxus, Branchiostoma lanceolatum [1]. Here we report on the complete amino acid sequence determination of 3 additional minor isoforms. The seven isoforms differ from each other in 9 positions of a contiguous 17-residue-long segment (positions 20-36) and can be classified in a alpha (ASCP I, III and IV) and a beta lineage (ASCP II, V, VI and VII).

Amino Acid Sequence↗

PubMed1992

Murine ST2 gene is a member of the primary response gene family induced by growth factors.

The murine ST2 gene, which encodes a protein remarkably similar to the extracellular portion of murine interleukin 1 receptor types 1 and 2, is expressed in growth-stimulated BALB/c-3T3 cells in the presence of 50 micrograms/ml of cycloheximide. The treatment with 1,000 U/ml of purified native murine beta-interferon superinduced, rather than suppressed, the ST2 mRNA expression as in the cases of c-myc and JE mRNAs. These results suggested that the murine ST2 gene belongs to the family of primary response genes induced by growth factors. Furthermore, a longer ST2-related mRNA was found in BALB/c-3T3 cells that were stimulated to proliferate in the presence of cycloheximide.

3T3 Cells↗

PubMed1992

Assessment study on the high-performance liquid chromatography-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate.

The HPLC-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate (SDS) was assessed with special attention to the behavior of the surfactant. A significant amount of SDS was found to be adsorbed to the hydroxyapatite packed in the column from the starting buffer, 50 mM sodium phosphate buffer, pH 7.0, only when the buffer contained SDS in a concentration at or above its critical micelle concentration. When the phosphate buffer concentration was increased while the SDS concentration was kept at 1 mg/ml, the adsorbed surfactant was desorbed in advance of the release of proteins. Polypeptides derived from proteins could be successfully separated only when the column had been thoroughly equilibrated with the above-mentioned starting buffer solution. When a protein polypeptide complexed with SDS, which had been similarly equilibrated, was applied to the column, an amount of SDS corresponding to 75-90% (w/w) of the surfactant originally bound to the polypeptide was released upon its binding to the hydroxyapatite. On the other hand, porin, an Escherichia coli outer membrane protein, retaining its trimeric native structure in the presence of SDS, released a significantly smaller amount of SDS. When the membrane protein was denatured to give a single polypeptide, it behaved in a manner similar to that of the other protein polypeptides. The mechanism of binding of the protein polypeptides was discussed on the basis of these results. The native and denatured entities of porin could be efficiently separated as the result of the difference in their mode of interaction with the hydroxyapatite.

Chromatography↗

PubMed1992

Simultaneous determination of a new gastrointestinal prokinetic agent (HSR-803) and its metabolites in human serum and urine by high-performance liquid chromatography using automated column-switching.

A method based on high-performance liquid chromatography using column-switching is described for the simultaneous determination of HSR-803 and its metabolites in human serum and urine. The system uses a six-port valve with a Nucleosil CN pre-column for on-line sample clean-up, and direct injection of samples. The limits of quantitation in serum and urine were 5 and 20 ng/ml for HSR-803 and 50 and 200 ng/ml for the metabolites, respectively. The coefficients of variation for the intra- and inter-day accuracies were between 0.8 and 7.1% for each compound. This method was applied to the pharmacokinetic studies in humans after oral administration of HSR-803.

Benzamides↗

PubMed1992

Interactions between anticollagen antibodies and chondrocytes.

OBJECTIVE: To investigate the interactions between anticollagen antibodies and living chondrocytes. METHODS: Mouse monoclonal anti-type II collagen (anti-CII) antibodies, rabbit anti-human CII, and rat anti-CII, anti-CIV, anti-CV, anti-CVI, and anti-CIX were studied in vitro to determine their ability to bind to the plasma membrane of living bovine chondrocytes. RESULTS: Mouse monoclonal anti-CII, rabbit anti-CII, and rat anti-CII, anti-CV, and anti-CIX were shown to bind in vitro to the plasma membrane of bovine chondrocytes. Antibody binding was not observed with anti-CIV, or with chondrocytes previously incubated with bacterial collagenase. A significant increase in chondrocyte caseinase and collagenase secretion was observed following sequential incubation with the monoclonal antibodies and a source of activating cytokines. CONCLUSION: These results suggest that collagen autoantibodies may exert some of their pathogenic effects on cartilage through interactions with resident chondrocytes, leading to modulation of the rate of secretion of cartilage matrix-degrading enzymes.

Animals↗

PubMed1992

Interactions of synovial fluid immunoglobulins with chondrocytes.

OBJECTIVE: To study the interaction of synovial fluid (SF) immunoglobulins with living chondrocytes, and to evaluate the relative contribution of type II collagen (CII) antibodies. METHODS: SF of patients with rheumatoid arthritis (RA), osteoarthritis (OA), and gout were incubated with isolated bovine articular chondrocytes. Ig binding was measured by flow cytometry and by quantitation with 125I-labeled anti-IgG and anti-IgM. Complement-dependent cytotoxicity was determined by 51Cr release. Immunoglobulin binding and cytotoxicity were compared between chondrocytes obtained from the superficial and from the deep cartilage zones. RESULTS: Significantly greater IgG and IgM binding was found with RA SF compared with OA or gout SF. Chondrocytes bound more Ig than did fibroblasts. The relative contribution of anti-CII antibodies to Ig binding was studied following absorption of the SF with bovine CII, and by incubation with bacterial collagenase-treated chondrocytes. There was a small but significant reduction in IgG and IgM binding with SF samples that were positive for anti-CII. RA SF exhibited modest, but significantly greater complement-dependent cytotoxicity than OA SF: Gel chromatography fractionation indicated that IgM antibodies were responsible for the cytotoxic activity. Additional studies showed that SF IgM antibodies bound preferentially to, and killed chondrocytes obtained from, the superficial layers of cartilage. CONCLUSION: Anti-CII antibodies contained in RA SF represent one of many antibody specificities reacting with chondrocyte membrane antigens. Chondrocyte-reactive SF antibodies may play an important pathogenic role in the processes leading to irreversible cartilage damage in RA. These deleterious effects appear to be exerted particularly on chondrocytes located near the articular surface of cartilage.

Animals↗

PubMed1992

The atypical M2 segment of the beta subunit confers picrotoxinin resistance to inhibitory glycine receptor channels.

Purified preparations of the inhibitory glycine receptor (GlyR) contain alpha and beta subunits, which share homologous primary structures and a common transmembrane topology with other members of the ligand-gated ion channel superfamily. Here, a beta subunit-specific antiserum was shown to precipitate the [3H]strychnine binding sites localized on alpha subunits from membrane extracts of both rat spinal cord and mammalian cells co-transfected with alpha and beta cDNAs. Further, inhibition of alpha homo-oligomeric GlyRs by picrotoxinin, a non-competitive blocker of ion flow, was reduced 50- to 200-fold for alpha/beta hetero-oligomeric receptors generated by cotransfection. Site-directed mutagenesis identified residues within the second predicted transmembrane segment (M2) of the beta subunit as major determinants of picrotoxinin resistance. These data implicate the M2 segment in blocker binding to and lining of the GlyR chloride channel.

Amino Acid Sequence↗

PubMed1992

Agarose for a bioartificial pancreas.

Islets were encapsulated into 5% concentration agarose microbeads. The effect of microencapsulation on islet allograft survivals was determined using a streptozotocin-induced diabetic (STZ) mouse and a nonobese diabetic (NOD) mouse as recipients. All five STZ BALB/c mice receiving microencapsulated islets (C57BL/6) maintained normoglycemia indefinitely. When NOD mice were used as recipients of the bioartificial pancreas, four of five grafts (islets from C3H/He) functioned for more than 80 d. Two of five NOD mice maintained normoglycemia until animals were sacrificed at 102 and 192 postoperative d. Microbeads made of commercially available agarose can effectively prolong alloislets functioning in the STZ-diabetic mouse and even in the NOD mouse (animal model of human type I diabetes) without the use of any immunosuppressive drug.

Animals↗

PubMed1992

Triplet pregnancy involving complete hydatidiform mole and two fetuses: genetic analysis by deoxyribonucleic acid fingerprint.

A case of a triplet pregnancy involving a dizygous twin pregnancy and a complete hydatidiform mole after therapy with human menopausal gonadotropin and human chorionic gonadotropin is reported. Two female fetuses, two placentas in one mass with two amnions and two chorions, and a tumor mass with a grapelike appearance were spontaneously delivered at 19 weeks of gestation. The deoxyribonucleic acid fingerprints of the two placentas and tumor tissue were compared with those of the parents. The fingerprints of the placentas showed patterns different from each other; however, all their polymorphic fragments could be traced back to either the father or mother. All polymorphic fragments of the tumor tissue were inherited only from the father (androgenesis). These results indicated that this triplet pregnancy involved a dizygous twin pregnancy and a complete hydatidiform mole.

Adult↗