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Biomedical subjects

T Takagi

Publications and source records attributed to T Takagi.

At least 415 records · Page 23Linked to original sources
PubMed1995

Effect of ethanol on DNA synthesis and c-met induction in Chang liver cells with long-term culture.

This investigation was performed to determine whether ethanol affects induction on Chang liver cells of the c-met protooncogene product (c-met), a cell-surface receptor for hepatocyte growth factor (HGF). These cells were cultured for 4 weeks with 5, 50, or 100 mmol/L ethanol, or 10, 100, or 200 mumol/L acetaldehyde. Ethanol was found to inhibit [3H]thymidine incorporation by cells in a dose-dependent manner. In addition, acetaldehyde at 100 mumol/L also inhibited [3H]thymidine uptake. Treatment with recombinant HGF led to enhanced [3H]thymidine incorporation by cells treated with ethanol or acetaldehyde as well as by those left untreated, with no significant differences in the rates of increase among these three cell groups. The amount of c-met messenger RNA, however, was unaffected by treatment with ethanol or acetaldehyde. Expression of c-met as measured by cell ELISA was also unchanged by both treatments. These results suggest that ethanol has no effect on c-met induction on Chang liver cells and that HGF may not be involved in the ethanol induced inhibition of DNA synthesis.

Cells, Cultured↗
PubMed1994

Drebrin, a development-associated brain protein from rat embryo, causes the dissociation of tropomyosin from actin filaments.

Drebrin is a development-associated neuroprotein whose cDNA into fibroblasts causes the formation of dendrite-like structures (Shirao, T., Kojima, N., and Obata, K. (1992) Neuroreport 3, 109-112). To explore molecular functions of drebrin during brain development, we purified drebrin from brains of rat embryos. Drebrin bound to actin filaments at a stoichiometry of 1:5 with a dissociation constant (Kd) of 1.2 x 10(-7) M. It strongly inhibited the actin binding activity of tropomyosin. Excess amounts of tropomyosin also inhibited the drebrin binding to actin filaments, suggesting that drebrin and tropomyosin competitively bind to actin filaments. Further, drebrin inhibited not only the actin binding activity of alpha-actinin but also the actin cross-linking activity of alpha-actinin. Gene transfection experiments revealed that tropomyosin was dissociated from actin filaments in drebrin-overexpressing fibroblasts. Thus we hypothesize that drebrin may destabilize actin filaments by dissociating tropomyosin and alpha-actinin from actin filaments, resulting in the formation of axon and dendrites during neuronal development.

Actin Cytoskeleton↗
PubMed1994

Primary structure and distribution of ryanodine-binding protein isoforms of the bullfrog skeletal muscle.

We have cloned two groups of cDNAs which encode isoforms of ryanodine-binding protein/Ca2+ release channel of the bullfrog skeletal muscle sarcoplasmic reticulum. One of the cDNA groups encodes the protein of 5,037 (or 5,031 with a deletion) amino acids with a molecular weight of 571,262 (or 570,607), which is identified as the alpha-isoform of the ryanodine-binding protein based on the amino acid sequence of three tryptic fragments of the purified protein. The other group of cDNAs encodes the protein of 4,868 amino acids with molecular weight of 553,029, which contains the sequences of three proteolytic fragments derived from the beta-isoform protein. About 70% of the amino acid sequence identity is present between alpha- and beta-isoforms of the bullfrog. The primary structure of the alpha-isoform is highly (80%) homologous to the ryanodine-binding protein cloned from rabbit skeletal muscle (type 1). The beta-isoform, on the other hand, is more than 85% identical with that from the rabbit brain (type 3), while it has only 67% overall identity with type 1. Analyses of RNA from various tissues of the bullfrog demonstrate that the beta-isoform is widely expressed, while the alpha-isoform is expressed mainly in skeletal muscle. A phylogenetic analysis of the ryanodine-binding protein/Ca2+ release channel family suggests that the various types of Ca2+ release channels have evolved from an ancestor gene. Possible differential roles of alpha- and beta-isoforms of ryanodine-binding protein in Ca2+ release mechanisms including skeletal muscle excitation-contraction coupling were discussed.

Animals↗
PubMed1994

The giant extracellular hemoglobin from the polychaete Neanthes diversicolor. The cDNA-derived amino acid sequence of linker chain L2 and the exon/intron boundary conserved in linker genes.

The 4000 kDa extracellular hemoglobin from the polychaete Neanthes diversicolor consists of three types of subunits; three 15 kDa monomers (chains M1, M2 and M3), a 45 kDa disulfide-bonded trimer of chains T1, T2 and T3, and two 50-55 kDa disulfide-bonded homodimeric linkers (chains L1 and L2). The latter linker subunits are essential for the assembly of the other heme-containing subunits, monomers and a trimer. The cDNA encoding the linker chain L2 was amplified by polymerase chain reaction (PCR), and the cDNA-derived amino acid sequence of 235 residues has been determined. The sequence showed 22-75% identity with other linker chains. All of the linker sequences examined so far have a highly conserved cysteine-rich segment at positions 89-130: Xaa3-Cys-Xaa6-Cys-Xaa6-Cys-Xaa6-Cys-Asp-Gly-X aa2-Asp-Cys-Xaa4-Asp-Glu-Xaa4-Cys, and the motif corresponds exactly to the cysteine-rich repeats of the ligand-binding domains of vertebrate low-density lipoprotein (LDL) receptors (Suzuki, T. and Riggs, A.F. (1993) J. Biol. Chem. 268, 13548-13555). A 287 bp intron interrupts the coding sequence of Neanthes L2 gene just at the N-terminal boundary of this motif, and the position of the splice junction was exactly conserved in Neanthes and Lumbricus linker genes. This suggests that the intron has been conserved for at least 450 million years in annelid linker genes. The evolutionary origin of the remaining parts of linker chains is unclear, but it is noteworthy that the topology of the two intrachain disulfide bridges in the C-terminal segment of linker chains is homologous with that of the carbohydrate-recognition domain of animal C-type lectin.

Amino Acid Sequence↗
PubMed1994

Characterization and primary structure of amphioxus troponin C.

Troponin C (TnC) from amphioxus (Protochordate) was purified and its primary structure determined. Unlike the case of vertebrates and other invertebrates, amphioxus TnC is found in the soluble fraction after extractions at physiological ionic strength in the presence of Ca2+. Edman sequencing combined with mass spectroscopy indicate that the protein contains 163 amino acid residues. It possesses an acetylated N-terminus (although a small percentage has a free Ser N-terminus) and either epsilon-N-methyllysine or epsilon-N-dimethyllysine in position 20. It displays about 50% sequence identity with vertebrate skeletal-muscle and cardiac-muscle TnC, 44% with TnC of sea squirt, also a Protochordate, and 30% with other invertebrate TnC. Like vertebrate TnC, amphioxus TnC contains a N-terminal alpha-helix plus the usual four ancestral Ca(2+)-binding regions, but analysis of the sequence suggests that the fourth site is not functional. Flow dialysis shows that amphioxus TnC binds three Ca2+ with the mean apparent affinity constant K' of 3.4 +/- 1.5 10(5) M-1. No cooperativity exists between the sites, and the presence of up to 10 mM Mg2+ does not influence the Ca(2+)-binding isotherm, indicating that the metal-binding sites are Ca(2+)-specific at physiological Mg2+ concentrations. It forms a Ca(2+)-dependent, 1:1 complex with melittin and rabbit or crayfish troponin I (TnI). Amphioxus TnC possesses one Trp residue in position 151 and one at the C-terminus. Trp fluorescence suggests that one or both residues are solvent-exposed in the metal-free form and efficiently shielded in the Ca2+ form. Although Mg2+ has no effect on the Ca2+ binding, the Trp fluorescence is influenced by millimolar Mg2+, suggesting the presence of one or more independent Mg(2+)-binding site(s). A phylogenetic analysis clearly shows that amphioxus TnC is positioned on the branch of the Chordates, but at a distance from the vertebrate TnC. Its place on the phylogenetic tree is in accordance with the consensus evolutionary phylogeny.

Amino Acid Sequence↗
PubMed1994

Purification, characterization and inhibition of dihydropyrimidinase from rat liver.

Dihydropyrimidinase (DHPase) was purified 564-fold over the initial rat liver extract, using heat, ammonium sulfate fractionation, DEAE-Sepharose CL-6B, carboxymethyl-Sepharose CL-6B, hydroxyapatite and Sephacryl S-300 chromatography. The purified enzyme was shown to be homogeneous by gel electrophoresis both in the presence and absence of SDS. Its molecular mass, determined by gel filtration, was 215 kDa and the subunit mass was 54 kDa. DHPase catalyzed the reversible cyclization of 5,6-dihydrouracil (H2Ura) to N-carbamoyl-beta-alanine or 5,6-dihydrothymine (H2Thy) to N-carbamoyl-beta-aminoisobutyric acid. Authentic 5-bromo-5,6-dihydrouracil (BrH2Ura) and commercially available H2Thy were racemic. However, these 5-substituted 5,6-dihydropyrimidines were hydrolyzed by over 96% and 98%, respectively, by DHPase. These results suggest that dihydropyrimidinase has no stereo specificities for 5-substituents of H2Ura. The addition of H2Ura and H2Thy competitively inhibited the enzyme activity against BrH2Ura. However, the addition of N-carbamoyl-beta-alanine or N-carbamoyl-beta-amino-isobutyric acid showed hyperbolic mixed-type inhibition, when BrH2Ura was used as the substrate. The values of the dissociation constants of BrH2Ura, N-carbamoyl-beta-alanine and N-carbamoyl-beta-aminoisobutyric acid were 17 microM, 0.38 mM and 0.38 mM, respectively. DHPase from the rat liver contains 4 mol Zn2+/mol active enzyme, presumably one atom/subunit. Zn2+ also inhibited the hydrolysis of BrH2Ura by the enzyme. The Ki for Zn2+ as an inhibitor of DHPase was 23 microM, and the maximum rate of inactivation was 0.057 min-1 at 37 degrees C. H2Ura and H2Thy protected the enzyme activity from Zn2+ inactivation.

Amidohydrolases↗
PubMed1994

Cerebellar stroke due to vertebral artery occlusion after cervical spine trauma. Two case reports.

The authors report two cases of cerebellar infarction due to vertebral artery (VA) occlusion resulting from cervical spine trauma. In one patient with dens and C2 body fracture, the left VA was occluded, resulting in a left cerebellar infarction. The second patient, with a subluxation of C4 on C5, presented with cerebellar swelling and infarction along with acute hydrocephalus secondary to bilateral vertebral artery occlusion. Because vertebral artery injuries with cervical trauma are rarely symptomatic, they can be easily overlooked. Bilateral or dominant vertebral artery occlusion, however, may cause rapid and fatal ischemic damage to the cerebellum and brain stem. Rapid recognition and optimal treatment for this injury depends on early vertebral artery angiography.

Angiography↗
PubMed1994

Size-dependent separation of proteins in the presence of sodium dodecyl sulfate and dextran in capillary electrophoresis: effect of molecular weight of dextran.

Dextran solutions are widely used as sieving medium in protein analysis by capillary electrophoresis in the presence of sodium dodecyl sulfate. We studied the effect of dextran molecular weight on the separation efficiency using different dextran preparations with wide and very narrow molecular weight distributions, in the range between 1270 and 2,000,000. Migration times and band broadening of proteins were significantly affected by the molecular weight of dextran. Migration times of proteins decreased as molecular weights of the dextrans decreased. Satisfactory separation of all the proteins was possible with all dextrans except those with molecular weights of 70,000 and 23,800 where bovine serum albumin and phosphorylase b failed to be separated. Unexpectedly rapid separation of all the proteins with enhanced resolution could be observed using two dextrans with a narrow molecular weight distribution, with molecular weights of 1270 and 5220. Clearly the use of dextran with higher molecular weight is not the only way to achieve efficient separation of proteins. The separation mechanism in the presence of the low molecular weight dextrans remains to be made clear in a future study.

Carbonic Anhydrases↗
PubMed1994

Histological changes of implanted collagen material during bone induction.

Studies were made on the fate of implanted material during bone induction. Mixtures of 1 mg of crude bone morphogenetic protein (BMP), or bovine serum albumin as a control, and 1.5 mg of bovine collagen, were pressed into discs and implanted under the fascia of the rectus abdominus muscle of rats. The tissues with implants were fixed 7, 10, and 14 days later and examined histologically. On day 7 after implantation, the implant was surrounded and invaded by alkaline phosphatase-positive cells. New bone and cartilage were seen at the periphery of the implant. In the regions of calcified cartilage and bone, these osteogenic matrices were intermixed with the implant. The mineral deposits were seen by electron microscopy not only on the osteogenic matrices but also on the implanted collagen. On day 14, the bone had spread to the center of the implant. No osteogenesis or chondrogenesis was seen in control implants. It was concluded that the calcification occurred on the implanted collagen during bone induction, and that it was related to successive bone formation and remodeling.

Alkaline Phosphatase↗
PubMed1994

Feasibility of agarose microbeads with xenogeneic islets as a bioartificial pancreas.

A bioartificial pancreas, that is, transplantation of islets of Langerhans (islets) which are enclosed in a semipermeable membrane, has been proposed as a treatment for type I diabetes. The islets are immuno-isolated from the host by the semipermeable membrane preventing rejection while maintaining control of glucose metabolism for an extended period. The purpose of the current research is to evaluate the feasibility of preparing agarose microbeads with xenogeneic hamster islets as a bioartificial pancreas in streptozotocin induced diabetic mice. In the recipients with a low level of anti-hamster antibodies, the combination of encapsulation of hamster islets in 5% agarose microbeads and in vitro culture of them prolonged xenograft survivals. Four of 6 recipients were still normoglycemic at 100 days after implantation. However, the same procedure was not effective in the recipients which were sensitized in advance by transplantation of free hamster islets and thus had high levels of anti-hamster antibodies. The average normoglycemic period was 32 days. Antibodies permeated through the microbeads and activated complement on the cell surfaces. The network of agarose microbeads was rendered dense by increasing the concentration of agarose to restrict the diffusion of antibodies. Graft survivals were prolonged with increasing concentrations of agarose. As an analysis using diffusion equations predicted, the survivals were inversely proportional to the diffusion coefficient of IgG in each agarose gel. Islet xenotransplantation was enabled by the combination of the microbeads with a concentration of agarose higher than 7.5% and in vitro culture even in recipients having a high level of preformed antibodies.

Animals↗
PubMed1994

Strategy for developing microbeads applicable to islet xenotransplantation into a spontaneous diabetic NOD mouse.

A bioartificial pancreas (BAP) created through the encapsulation of islets of Langerhans (islets) in a semipermeable membrane has been proposed as a promising approach to treating insulin-dependent diabetes patients. A nonobese diabetic (NOD) mouse, which shares many features of human insulin-dependent diabetes mellitus, is an ideal model for evaluating the function of BAP. However, the functions of BAPs that have been developed have been limited in NOD mice. We propose novel microbeads that can realize long-term BAP function in NOD mice. The novel microbeads were composed of agarose and poly(styrene sulfonic acid) (PSSa) mixed gel. A polyion complex layer between PSSa and polycationic polybrene was formed on and just inside the microbead, and the microbead surfaces were further covered by polyanions to produce anionic surface charges. The islets in the novel microbeads were intraperitoneally implanted. Graft-functioning periods were dependent on both PSSa concentration and the kinds of polyanion. Islets in the microbeads composed of 5% agarose and 5% PSSa, which had an outermost surface covered by carboxymethyl cellulose, produced normoglycemic periods of more than 60 days in all five recipients. Control mice receiving either transplants of unenclosed islets or islets in agarose microbeads showed normoglycemic periods of less than 12 days. We believe that agarose/PSSa microbeads are promising for producing semipermeable membranes that enable xenotransplantation of islets in spontaneous diabetes mellitus.

Animals↗
PubMed1994

A deductive database system for analyzing human nucleotide sequence data.

The analysis of the human genome is one of the most significant topics in both biology and medical science. There is a growing need for a well-designed database system for searching and analyzing the human genome data. We developed a deductive database system to search and analyze nucleotide sequence data derived from the GenBank primates data. A deductive database system is a next generation one and it contains an inference mechanism that can handle problems beyond the capabilities of classical database systems. Database queries are described in logical rules. These rules are simple even for molecular biologists who are not experts in computer programs because they are declarative and do not require the procedural commands that are usually used in computer programs. Furthermore, queries based on logical rules are powerful enough to express complicated biological problems. Particularly, recursive rules are suitable for examining secondary structures of nucleotide sequences. In our analysis of TfR's IRE, we noted five stem-and-loop structures.

Artificial Intelligence↗
PubMed1994

Comparative studies of in vitro and in vivo function of three different shaped bioartificial pancreases made of agarose hydrogel.

Various kinds of bioartificial pancreas (BAP) have been developed in the past. There have been many disputes about the advantages and disadvantages of each BAP. However, little attention has been paid to the shape of the devices. In this study, three different shaped BAPs were made of the same material, agarose hydrogel. These are microbead-, rod- and disc-shaped BAPs, which are comparable to microcapsules, hollow fibre diffusion chambers and disc-shaped diffusion chambers, respectively, in shape. Their performances were studied by in vitro culture, mathematical simulations and in vivo implantation into diabetic animals. The islets in the BAPs could survive and release insulin for more than 100 d regardless of the BAP shape during in vitro culture. Numerical analyses showed that insulin release kinetics depended greatly on the thickness of the gel membrane but not on the shape of the agarose hydrogel. The microbead BAP normalized blood glucose levels of all five recipients for more than 100 d. Three of eight recipients of the rod BAP and only one of six recipients of the disc BAP demonstrated long normoglycaemia. These results suggest that the shape of the BAPs determines the in vivo functioning period of the BAPs and that the microbead is the most suitable shape for the BAP.

Animals↗
PubMed1994

Differential expression of sgk mRNA, a member of the Ser/Thr protein kinase gene family, in rat brain after CNS injury.

We cloned genes the expression of which were induced 3 days after cortical injury of rat brain by a differential display technique, and four novel and known sequences were isolated. Among these sequences, the sgk gene which was recently identified as a novel member of the serine/threonine protein kinase gene family, was selected for analysis of its expression patterns in rat brain by northern blotting and in situ hybridization, because hybridization signals were strong at the lesion sites. Expression of sgk mRNA was induced within 3 days after injury, and was maintained at a high level for at least 14 days. The cells which strongly expressed the sgk gene were in the deep layers of the cortex and in the corpus callosum. In situ hybridization analysis for sgk and myelin proteolipid protein mRNA using serial sections showed that the distribution of both signals was very similar at the damaged regions. Therefore, it is likely that the sgk transcript is expressed by oligodendrocytes after brain injury. Investigation of the developmental expression of the sgk gene showed that neurons in layers I and II of the cortex, lateroposterior and laterodorsal thalamic nucleus, and ventral posterolateral and posteromedial thalamic nucleus strongly expressed sgk mRNA at postnatal day 1 and day 7, but these neurons showed no expression in fetal or adult brain. These results suggest that the induction of sgk gene may be associated with a series of axonal regenerations after brain injury, and in addition, the sgk gene may also play important roles in the development of particular groups of neurons in the postnatal brain.

Aging↗
PubMed1994

Cloning and expression of a neural differentiation-associated gene, p205, in the embryonal carcinoma cell line P19 and in the developing mouse.

Mouse P19 embryonal carcinoma cells can be reproducibly differentiated into neurons and glial cells upon treatment with high concentration of retinoic acid (RA). In order to understand the molecular mechanisms that control early neural differentiation, we screened a cDNA library made from 24-h RA-treated P19 cells with subtracted cDNA probes. One clone was positive in the secondary screening and was designated as p205. This clone (1.1 kb) has an open reading frame of 317 amino acids with homology to G-protein beta subunit. This protein sequence was identical to chicken and human genes previously identified as a major histocompatibility complex-associated gene. The complete conservation of its amino acid sequence between mouse, human and chicken provides strong evidence that the p205 protein fulfills a fundamental function. Developmental Northern blot analysis revealed that a p205 mRNA is expressed at high levels in the embryonic mouse brain, decreasing as development proceeds. In situ hybridization revealed that p205 mRNA is strongly and ubiquitously expressed in the embryonic and early postnatal mouse brain. This expression decreased during postnatal development and was localized in the dentate gyrus, habenula, piriform cortex, paraventricular nucleus of the hypothalamus and supraoptic nucleus of the adult brain. These results suggest that this protein plays an important role in the developing brain and neuronal differentiation.

Aging↗
PubMed1994

Simulation study on the effect of external vibration on left ventricular function: potential indicator of LV tolerance to the sudden reduction of myocardial contractility.

In a previous study the authors reported that external mechanical vibration applied to the left ventricular (LV) epicardium induces contractility-dependent depression in LV pressure, stroke volume and stroke work. It was suggested that this depression may be caused by the direct effect of external vibration on contractile protein. In another paper in this issue, it is proved that LV function with various myocardial contractilities and the actual process of deterioration in heart failure are well simulated in the model proposed by Beyar and Sideman, after some modifications have been made. In the study reported here it is assumed that an external mechanical vibration induces sudden reduction in myocardial active stress in the model of Beyar and Sideman; in this way the contractility-dependent effect of external vibration on LV function has been simulated. The results of this simulation support the suggestion that external mechanical vibration directly affects contractile protein and reduces LV function, and it is further suggested that the reduction of LV function induced by external vibration reflects the reserve or tolerance capacity of LV to a sudden reduction of myocardial contractility.

Animals↗
PubMed1994

Fusion of phospholipid vesicles induced by clathrin: modulation of fused liposome size.

The effects of lipid composition, temperature, and ionic strength on critical events in the membrane fusion reaction induced by clathrin, such as membrane binding and self-association of clathrin, membrane aggregation, and actual fusion, and the size of fused liposomes, were examined using large unilamellar vesicles (LUV) containing acidic phospholipids. When membrane fusion and aggregation of LUV with different lipid compositions were initiated at 25 degrees C in 0.1 M NaCl by lowering the pH of the medium from 7.40 to 4.75 in the presence of clathrin, two types of reaction processes were observed: fusion and aggregation of LUV containing phosphatidylserine and phosphatidic acid occurred slowly with a long fusion lag-phase and reached a high level, whereas the two reactions of LUV containing phosphatidylglycerol and phosphatidylinositol were induced faster with a much shorter fusion lag-time and leveled off in a shorter time. Similar differences in the fusion reactions were observed in media at different temperatures and ionic strengths with any type of LUV: slow and extensive aggregation and fusion occurred at low temperature and/or high ionic strength, but faster, less extensive reactions occurred at high temperature and/or low ionic strength, indicating that the two types of reaction pattern are due to the dependencies of the aggregation and fusion reactions on the temperature and ionic strength, but not on the lipid composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Clathrin↗